New imidazo(1,2-a)pyrazine derivatives with bronchodilatory and cyclic nucleotide phosphodiesterase inhibitory activities.

New imidazo[1,2-a]pyrazine derivatives have been synthesized either by direct cyclization from pyrazines or by electrophilic substitutions. The presence of electron donating groups on position 8 greatly enhances the reactivity of the heterocycle towards such reactions on position 3 of the heterocycle. The activities of these derivatives in trachealis muscle relaxation and in inhibiting cyclic nucleotide phosphodiesterase (PDE) isoenzyme types III and IV have been assessed. All compounds demonstrated significantly higher relaxant potency than theophylline. All the derivatives were moderately potent in inhibiting the type IV isoenzyme of PDE but only those with a cyano group on position 2 were potent in inhibiting the type III isoenzyme.

Effects of SCA40 on bovine trachealis muscle and on cyclic nucleotide phosphodiesterases.

While UK-93,928 (1-[[3-(6,9-dihydro-6-oxo-9-propyl-1H-purin-2-yl)-4-ethoxyphenyl] sulfonyl]-4-methylpiperazine; 5 nM-5 microM) was devoid of relaxant activity, benzafentrine, isoprenaline, levcromakalim and SCA40 (6-bromo-8-methylaminoimidazo[1,2-a]pyrazine-2-carbonitrile) each relaxed histamine (460 microM)-precontracted bovine isolated trachealis. Each of these relaxants was antagonised by a K+-rich (80 mM) medium. Except in the case of levcromakalim, nifedipine (1 microM) offset this antagonism. Charybdotoxin (100 nM) antagonised isoprenaline in a nifedipine-sensitive manner but did not antagonise SCA40 or benzafentrine. Iberiotoxin (100 nM) did not antagonise SCA40. Acting on tissue precontracted with carbachol, SCA40 potentiated isoprenaline but did not potentiate sodium nitroprusside. While levcromakalim (1 and 10 microM) induced hyperpolarisation, SCA40 (1 and 10 microM) induced little change in the membrane potential of bovine trachealis. In trachealis preloaded with 86Rb+, levcromakalim (1 and 10 microM) promoted efflux of the radiotracer while SCA40 (1 and 10 microM) had no effect. Tested as an inhibitor of isoenzymes of cyclic nucleotide phosphodiesterase, SCA40 was most potent against the type III, less potent against the type IV and least potent against the type I isoenzyme. It is concluded that neither inhibition of phosphodiesterase type V nor the promotion of BKCa channel opening explains the tracheal smooth muscle relaxant activity of SCA40. This compound relaxes bovine tracheal smooth muscle mainly by inhibiting phosphodiesterase isoenzyme types III and IV.

The spasmogenic effects of vanadate in human isolated bronchus.

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 microM-3 mM) produced concentration-dependent, well-sustained contraction. Its -logEC50 was 3.74 +/- 0.05 (mean +/- s.e.mean) and its maximal effect was equivalent to 97.5 +/- 4.2% of the response to acetylcholine (ACh, 1 mM). 2. Vanadate (200 microM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 microM), zileuton (10 microM), a mixture of atropine, mepyramine and phentolamine (each at 1 microM), or by mast cell degranulation with compound 48/80. 3. Vanadate (200 microM)-induced contraction was unaltered by tissue exposure to verapamil or nifedipine (each 1 microM) or to a Ca2+-free, EGTA (0.1 mM)-containing physiological salt solution (PSS). However, tissue incubation with ryanodine (10 microM) in Ca2+-free, EGTA (0.1 mM)-containing PSS reduced vanadate-induced contraction. A series of vanadate challenges was made in tissues exposed to Ca2+-free EGTA (0.1 mM)-containing PSS with the object of depleting intracellular Ca2+ stores. In such tissues cyclopiazonic acid (CPA; 10 microM) prevented Ca2+-induced recovery of vanadate-induced contraction. 4. Tissue incubation in K+-rich (80 mM) PSS, K+-free PSS, or PSS containing ouabain (10 microM) did not alter vanadate (200 microM)-induced contraction. Ouabain (10 microM) abolished the K+-induced relaxation of human bronchus bathed in K+-free PSS. This action was not shared by vanadate (200 microM). The tissue content of Na+ was increased and the tissue content of K+ was decreased by ouabain (10 microM). In contrast, vanadate (200 microM) did not alter the tissue content of these ions. Tissue incubation in a Na+-deficient (25 mM) PSS or in PSS containing amiloride (0.1 mM) markedly inhibited the spasmogenic effect of vanadate (200 microM). 5. Vanadate (200 microM)-induced contractions were markedly reduced by tissue treatment with each of the protein kinase C (PKC) inhibitors H-7 (10 microM), staurosporine (1 microM) and calphostin C (1 microM). Genistein (100 microM), an inhibitor of protein tyrosine kinase, also reduced the response to vanadate. 6 Vanadate (0.1-3 mM) and ACh (1 microM- 3 mM) each increased inositol phosphate accumulation in bronchus. Such responses were unaffected by a Ca2+-free medium either alone or in combination with ryanodine (10 microM). 7. In human cultured tracheal smooth muscle cells, histamine (100 microM) and vanadate (200 microM) each produced a transient increase in intracellular Ca2+ concentration ([Ca2+]i). 8. Intracellular microelectrode recording showed that the contractile effect of vanadate (200 microM) in human bronchus was associated with cellular depolarization. 9. It is concluded that vanadate acts directly on human bronchial smooth muscle, promoting the release of Ca2+ from an intracellular store. The Ca2+ release mechanism involves both the production of inositol phosphate second messengers and inhibition of Ca-ATPase. The activation of PKC plays an important role in mediating vanadate-induced contraction at values of [Ca2+]i that are close to basal.

Effects of some K(+)-channel inhibitors on the electrical behaviour of guinea-pig isolated trachealis and on its responses to spasmogenic drugs.

1. A study has been made of the effects of inhibitors selective among plasmalemmal K(+)-channels on the sensitivity and responsiveness of guinea-pig trachealis muscle to carbachol, histamine and KCl. The effects of the K(+)-channel inhibitors on the resting membrane potential and spontaneous electrical activity of the trachealis cells have also been examined. 2. In indomethacin (2.8 microM)-treated trachealis muscle, dofetilide (1 microM) and glibenclamide (10 microM) were each devoid of spasmogenic activity. In contrast, 4-aminopyridine (4-AP, 62.5 microM--8 mM), charybdotoxin (ChTX, 100 nM) and iberiotoxin (IbTX, 100 nM) were each spasmogenic. Spasm evoked by 4-AP, IbTX or ChTX was reduced, though not abolished, by atropine (1 microM). Spasm evoked by 4-AP (1 mM), ChTX (100 nM) or IbTX (100 nM) was unaffected by tetrodotoxin (TTX; 3.1 microM) or by tissue pretreatment with capsaicin (1 microM for 30 min). Spasm evoked by IbTX or ChTX was abolished by nifedipine (1 microM). 3. Dofetilide (1 microM) and glibenclamide (10 microM) were each without effect on the tracheal sensitivity or responsiveness to carbachol, histamine or KCl. 4-AP (1 mM) antagonized carbachol, potentiated histamine but did not affect tissue sensitivity to KCl. When the effects of 4-AP were examined in the presence of atropine (1 microM), it potentiated all the spasmogens including carbachol. IbTX and ChTX (each 100 nM) potentiated all three spasmogens. Potentiation of histamine induced by 4-AP (1 mM) or IbTX (100 nM) was also observed in tissues treated with a combination of atropine (1 microM) and TTX (3.1 microM). 4. Dofetilide (1 and 10 microM) was without effect on the resting membrane potential or spontaneous electrical activity of the trachealis cells. 4-AP (1 mM) evoked depolarization and caused a small increase in the frequency of slow wave discharge. The depolarization evoked by 4-AP was abolished by atropine (1 microM). IbTX (100 nM) and ChTX (100 nM) each evoked little or no change in resting membrane potential but converted the spontaneous slow waves into spike-like, regenerative action potentials. These electrophysiological effects of IbTX and ChTX were unaffected by atropine (1 microM). 5. It is concluded that the dofetilide-sensitive, cardiac, delayed rectifier K(+)-channel is either not expressed in trachealis muscle or is of no functional importance in that tissue. The ATP-sensitive K(+)-channel (KATP) does not moderate tracheal sensitivity to spasmogens such as carbachol, histamine and KCl. The 4-AP-sensitive delayed rectifier K(+)-channel (Kdr) and the large Ca(2+)-dependent K(+)-channel (BKCa) each moderate trachealis muscle sensitivity to spasmogens. Neither Kdr nor BKCa plays an important role in determining the resting membrane potential of guinea-pig trachealis cells. However, the BKCa channel is responsible for limiting the effects of the increase in membrane Ca2+ conductance associated with the depolarizing phase of slow waves. It is BKCa channel opening that prevents the development of a slow wave into a spike-like regenerative action potential.

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. 6. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of SCA40 (1 micro M) were often accompanied by the suppression of spontaneous electrical slow waves but no change in resting membrane potential. When the concentration of SCA40 was raised to 10 micro M, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization.7. SCA40 (10 nM- 100 micro M) more potently inhibited the activity of cyclic AMP phosphodiesterase (PDE)than that of cyclic GMP PDE derived from homogenates of guinea-pig trachealis. Theophylline(1 micro M- 1O mM) also inhibited these enzymes but was less potent than SCA40 in each case and did not exhibit selectivity for inhibition of cyclic AMP hydrolysis.8. Tested against the activity of the isoenzymes of cyclic nucleotide PDE derived from human blood cells and lung tissue, SCA40 proved highly potent against the type III isoenzyme. It was markedly less potent against the type IV and type V isoenzymes and even less potent against the isoenzymes types I and II.9. It is concluded that the tracheal relaxant action of SCA40 (1 nM- 1 micro M) does not involve the activation of beta-adrenoceptors or P1 or P2 purinoceptors. Furthermore, this action is unlikely to depend upon the opening of BKca channels with consequent cellular hyperpolarization and voltage-dependent inhibition of Ca2+ influx. The tracheal relaxant action of SCA40 (up to 1 micro M) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 micro M, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKca channels.

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). 6. Tested in trachea bathed by K+-rich (40 mM), Ca2+-free PSS, CaCl2 (0.1-20 mM) caused concentration-dependent spasm. PDA (1-5 MicroM) did not significantly modify the shape or position of the log concentration-effect curve for CaCl2. In contrast, verapamil (1 and 10 MicroM) antagonized CaCl2.7. Tested in trachea bathed by indomethacin (2.8 MicroM)-containing PSS, ryanodine (1-100 MicroM) caused concentration-dependent spasm. PDA (5 MicroM) potentiated ryanodine. Verapamil (10 MicroM) inhibited ryanodine in inducing spasm and suppressed the ability of PDA to potentiate ryanodine.8. It is concluded that, in guinea-pig isolated trachea, PDA augments the spasmogenic activity of the methylxanthines and ryanodine. This effect of PDA does not result from PDA-induced suppression of spontaneous tone, from increased cellular entry of Ca2+ through L-type channels or from sensitization of the intracellular contractile machinery to activator Ca2+. The evidence suggests, instead, that PDA facilitates methylxanthine- or ryanodine-induced release of Ca2+ from the intracellular store.

Airway response to inhaled methacholine in normal human subjects.

The individual airway responsiveness to inhaled, nebulized methacholine (MeCh) was estimated in normal volunteers, measuring specific airway conductance (sGAW). The dose of MeCh was increased logarithmically until a 60-65% reduction from baseline sGAW or an asymptotic approach to a maximal response was attained. The concentration of MeCh that caused a 35% reduction in sGAW (PC35), the dose that caused a 62.5% reduction in sGAW, the slope of the straight, central part of the log-dose-response curve (LDRC), the slope of the straight, initial part of the dose-response curve, the maximal response attainable (Emax) and the dose causing a half-maximal response (ED50) were derived. These parameters were transformed as necessary to attain normality of distribution. Relationships between them were examined by measuring the correlations between their transformed values. The ED50 was taken to represent the least biased estimate of the sensitivity to MeCh. The PC35 was the best practical estimate of sensitivity. The Emax was taken to represent the least biased estimate of the reactivity to MeCh. The slope of the LDRC was the best practical estimate of reactivity. The sensitivity and reactivity varied independently in these normal subjects. Each was also independent of the baseline sGAW.

Beta-adrenoceptor subtypes and the opening of plasmalemmal K(+)-channels in trachealis muscle: electrophysiological and mechanical studies in guinea-pig tissue.

1. Mechanical and electrophysiological studies of guinea-pig isolated trachealis have been made with the objectives of: (a) identifying which of the beta-adrenoceptor subtypes mediates the opening of plasmalemmal K(+)-channels, (b) gaining further insight into the properties of the novel, long-acting beta-adrenoceptor agonist, salmeterol and (c) clarifying the role of K(+)-channel opening in mediating the relaxant actions of agonists at beta-adrenoceptors. 2. Noradrenaline (10 nM-100 microM) caused a concentration-dependent increase in the rate of beating of guinea-pig isolated atria. The selective beta 1-adrenoceptor blocking drug, CGP 20712A (100 nM-10 microM) caused concentration-dependent antagonism of noradrenaline. The selective beta 2-adrenoceptor blocking drug, ICI 118551, also produced concentration-dependent antagonism of noradrenaline, but only when used in concentrations greater than 300 nM. 3. Cromakalim (100 nM-10 microM), isoprenaline (1-100 nM), procaterol (0.1-30 nM), salbutamol (1 nM-1 microM), salmeterol (1-100 nM) and theophylline (1 microM-1 mM) each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. 4. ICI 118551 (10 nM-1 microM) antagonized isoprenaline, procaterol and salmeterol in suppressing the spontaneous tone of the isolated trachea. The antagonism was concentration-dependent. In contrast, ICI 118551 (1 microM) antagonized neither cromakalim nor theophylline. CGP 20712A (up to 1 microM) failed to antagonize cromakalim, isoprenaline, procaterol, salmeterol or theophylline. In trachea treated with indomethacin (2.8 microM) and carbachol (10 microM), salmeterol (1 microM) antagonized the effects of isoprenaline but not aminophylline. 5. Intracellular electrophysiological recording from guinea-pig isolated trachealis showed that the relaxant effects of cromakalim (10 microM), isoprenaline (100 nM), procaterol (10 nM) and salbutamol(10 nM- 1 microM) were accompanied by the suppression of spontaneous electrical slow waves and by cellular hyperpolarization. In contrast, the relaxant effects of salmeterol (10 nM- 1 microM) were not accompanied by significant cellular hyperpolarization.6. CGP 20712A (1 microM) inhibited the hyperpolarization but not the relaxation induced by isoprenaline(100 nM). In contrast ICI 118551 (100 nM) inhibited both the hyperpolarization and the relaxation induced by isoprenaline (100 nM). Neither CGP 20712A (1 microM) nor ICI 118551 (100 nM) inhibited the hyperpolarization induced by cromakalim (10 microM). Salmeterol (1 microM) inhibited the hyperpolarization induced by isoprenaline (100 nM) but not that induced by cromakalim (10 microM).7. It is concluded that activation of either beta l- or beta 2-adrenoceptors can promote the opening of K+-channels in the trachealis plasmalemma. The poor ability of salmeterol to hyperpolarize trachealis muscle reflects neither its selectivity in activating beta 2-adrenoceptors as opposed to beta 1-adrenoceptors nor a non-specific action in stabilizing the cell membrane. Instead, it may reflect low intrinsic efficacy of the drug at beta 2-adrenoceptors. The opening of plasmalemmal K+-channels plays a supportive rather than a crucial role in mediating the tracheal relaxant actions of agonists at beta-adrenoceptors.

Beta-adrenoceptor subtypes and the opening of plasmalemmal K(+)-channels in bovine trachealis muscle: studies of mechanical activity and ion fluxes.

1. Studies of mechanical activity and 86Rb+ efflux have been made in bovine isolated trachealis with the objectives of: (a) identifying which of the beta-adrenoceptor subtypes mediates the opening of plasmalemmal K(+)-channels, (b) gaining further insight into the properties of the novel, long-acting beta 2-adrenoceptor agonist, salmeterol and (c) clarifying the role of K(+)-channel opening in mediating the mechano-inhibitory actions of agonists at beta-adrenoceptors. 2. In bovine trachealis muscle strips precontracted with histamine (460 microM), isoprenaline (0.1 nM-1 microM), procaterol (0.1-10 nM) and salmeterol (0.1-10 nM) each caused concentration-dependent relaxation. 3. ICI 118551 (10 nM-1 microM) antagonized isoprenaline, procaterol and salmeterol in suppressing histamine-induced tone of the isolated trachealis muscle. The antagonism was concentration-dependent. In contrast, CGP 20712A (10 nM-1 microM) failed to antagonize isoprenaline, procaterol or salmeterol. 4. Salmeterol (1-10 microM) antagonized isoprenaline in relaxing strips of bovine trachea which had been precontracted with carbachol (1 microM). 5. Cromakalim (10 microM), isoprenaline (100 nM-10 microM), procaterol (10 nM-1 microM) and salbutamol (100 nM-10 microM) each promoted the efflux of 86Rb+ from strips of bovine trachealis muscle preloaded with the radiotracer. In contrast, salmeterol (100 nM-10 microM) failed to promote 86Rb+ efflux. 6. CGP 201712A (1 microM), ICI 118551 (100 nM) and salmeterol (1 microM) did not themselves modify 86Rb+ efflux from trachealis muscle strips, nor did they affect the promotion of 86Rb+ efflux induced by cromakalim (10 microM). In contrast, CGP 20712A (1 microM) and ICI 118551 (100nM) were each able to inhibit the promotion of 86Rb+ efflux induced by isoprenaline (1 microM) or procaterol (100 nM). Furthermore,salmeterol (10 microM) inhibited isoprenaline (1 microM)-induced promotion of 86Rb+ efflux.7. It is concluded that, in bovine trachealis, activation of either beta l- or beta 2-adrenoceptors can promote the opening of 86Rb+-permeable K+-channels in the plasmalemma. The failure of salmeterol to promote plasmalemmal K+-channel opening may reflect, not its selectivity in activating beta 2- as opposed to beta 1-adrenoceptors, but rather its low intrinsic efficacy at beta 2-adrenoceptors. The opening of plasmalemmal K+-channels plays a supportive rather than a crucial role in mediating the mechano-inhibitory effects of agonists at beta-adrenoceptors acting on trachealis muscle.

The properties of voltage-operated Ca(2+)-channels in bovine isolated trachealis cells.

Freshly-dispersed bovine trachealis cells were used for recording by the patch clamp technique of whole-cell and unitary currents through Ca(2+)-channels. Whole-cell Ca(2+)-current (ICa) activated at -40 mV and appeared to be carried by a single type of Ca(2+)-channel. Inactivation of ICa was increased by increasing the concentration of free Ca2+ within the recording pipette but reduced by using Ba2- as the charge carrier. Steady-state inactivation studies showed that the Ca(2+)-channels were half-maximally available following a conditioning depolarization to -35 mV. A two-pulse protocol showed that ICa induced by the step to a test potential was inversely related to ICa induced by the step to the conditioning potential. Unitary Ba(2+)-currents were activated at a threshold of -30 mV and had a reversal potential of +41 mV. The channel carrying the unitary Ba(2+)-currents had a slope conductance of 23 pS. Steady-state inactivation studies showed that the unitary Ba(2+)-currents were half-maximally available at a holding potential of -28 mV. ICa and unitary Ba(2+)-currents were inhibited by nifedipine (10 nM-1 microM) but augmented by Bay K 8644 (10 microM). It is concluded that the plasmalemma of bovine trachealis muscle contains a single population of voltage-dependent Ca(2+)-channels of the L-type. These channels may be subject to inactivation primarily by an increase in the concentration of free Ca2+ on the cytosolic side of the plasmalemma and secondarily by a voltage-dependent mechanism. Overlap of the inactivation and activation curves of ICa may allow the passage of 'window current' through the Ca(2+)-channels during sustained depolarization.

Trials of the bronchodilator activity of the xanthine analogue SDZ MKS 492 in healthy volunteers during a methacholine challenge test.

An approximately steady-state reduction of specific airway conductance was induced in healthy human subjects by means of an individualized inhaled methacholine loading dose followed by a maintenance dose regime. Tested against this background bronchoconstriction, the xanthine analogue SDZ MKS 492, when administered as a single oral dose of 40 mg, showed a significant bronchodilator action, which lasted for up to 5.5 h. Bronchodilatation was not seen after administration of 10 or 20 mg doses. SDZ MKS 492 inhaled as a dry powder had a bronchodilator action that was small, most evident with the 12 mg dose and transient. The peak relief of imposed bronchoconstriction was 29% and the apparent half-time of removal of SDZ MKS 492 from its site of action was 5-6 min. Inhaled SDZ 492 had a bitter taste that was not masked by inclusion of menthol and aspartame in the formulation. The bronchodilatation seen in laboratory animals can also be produced by SDZ MKS 492 in man when administered orally or by inhalation. Its magnitude correlates better with the plasma concentration of parent drug than with that of either of the identified metabolites. Dispositional processes in the lung abbreviate its action after administration by inhalation.

Trials of the bronchodilator activity of the isoenzyme-selective phosphodiesterase inhibitor AH 21-132 in healthy volunteers during a methacholine challenge test.

1. An approximately steady-state reduction of specific airway conductance was induced in normal human subjects by means of a methacholine individualized loading+maintenance dose regime. Tested against this background bronchoconstriction, the mixed type III/IV phosphodiesterase inhibitor AH 21-132, ingested in doses up to 90 mg, had no detectable bronchodilator activity. 2. AH 21-132, infused intravenously over 15 min, evoked short-lived bronchodilatation at doses of 20 and 40 mg, without affecting blood pressure or heart rate. 3. AH 21-132, mixed 1:18.5 by weight with sucrose, dissolved in saline, nebulized and inhaled in doses between 2 and 24 mg of AH 21-132, produced dose-dependent bronchodilation. The ED50 was estimated as 9.2 mg AH 21-132. The peak relief of imposed bronchoconstriction was 80% and the apparent half-time of removal of AH 21-132 from its site of action was 25 min. 4. Inhaled, nebulized, hypertonic sucrose had a minor bronchodilator action. 5. AH 21-132, by intravenous and inhaled routes of administration, provides relief of methacholine-induced bronchoconstriction.

Inhibition by adrenergic neurone blocking agents of the relaxation induced by BRL 38227 in vascular, intestinal and uterine smooth muscle.

1. The adrenergic neurone blocking agents, guanethidine and bretylium, have been tested for inhibitory activity against the actions of some relaxant drugs (BRL 38227, noradrenaline, sodium nitroprusside, theophylline) in vascular, intestinal and uterine smooth muscle. 2. In guinea-pig isolated taenia caeci pre-contracted with KCl (25 mM), BRL 38227 (0.1-10 microM) and noradrenaline (10 nM-100 microM) each caused concentration-dependent relaxation. Guanethidine and bretylium (50 microM) each antagonized the relaxation to BRL 38227 but not that to noradrenaline. At high concentration (500 microM), the adrenergic neurone blocking agents antagonized the action of BRL 38227 and, to some extent, that of noradrenaline. 3. In rat isolated aorta pre-contracted with noradrenaline (300 nM), BRL 38227 (0.0125-3.2 microM) and sodium nitroprusside (0.3-100 nM) each produced concentration-dependent smooth muscle relaxation. Guanethidine and bretylium (5-500 microM) each antagonized the action of BRL 38227 without antagonizing that of sodium nitroprusside. 4. Rats were pretreated with 17-beta oestradiol benzoate. Tension waves were then induced from segments of isolated, oestrogen-dominated uterus by transmural electrical stimulation or by oxytocin (0.2 nM). These tension waves were inhibited by BRL 38227 (0.025-3.2 microM) or theophylline (0.05-0.8 mM) in a concentration-dependent manner. Guanethidine (50 microM) antagonized the action of BRL 38227 in both the electrically- and oxytocin-driven tissues. In the electrically-driven tissues, guanethidine (50 microM) did not antagonize the inhibition to theophylline. 5. In KCl (25 mM)-treated guinea-pig taenia caeci, guanethidine (50 microM) inhibited the efflux of 86Rb+ evoked by BRL 38227 (10 microM) but not that evoked by noradrenaline (10 microM). In contrast, apamin(100 nM) reduced the efflux of 86Rb+ which was promoted by noradrenaline, but did not affect efflux induced by BRL 38227.6. It is concluded that the adrenergic neurone blocking agents, guanethidine and bretylium (each at 50 microM), selectively inhibit the relaxant action of BRL 38227 in vascular, intestinal and uterine smooth muscle. If this inhibition reflects direct blockade of the K+-channel (KKCO) which is opened by BRL 38227, then the adrenergic neurone blocking agents act as inhibitors selective for KKCO as opposed to the small, apamin-sensitive (SKCa) and large (BKca) conductance, Ca2"-dependent K+-channels.

Analysis of the relaxant action of SDZ PCO 400 in airway smooth muscle from the ox and guinea-pig.

SDZ PCO 400 (30 nM-100 microM) suppressed the spontaneous tone of guinea-pig isolated trachealis. Glibenclamide (1-10 microM), phentolamine (100 microM), guanethidine (50 microM) and bretylium (50 microM) each antagonized SDZ PCO 400 without antagonizing isoprenaline or theophylline. Charybdotoxin (100 nM) failed to antagonize SDZ PCO 400 but antagonized theophylline. The relaxant action of SDZ PCO 400 was ablated when spasm was induced by a K(+)-rich (120 mM) medium. In bovine and guinea-pig trachea, SDZ PCO 400 (10 microM) suppressed spasm evoked by lower (less than 40 mM) but not higher (greater than 40 mM) concentrations of KCl. In guinea-pig trachea the relaxant action of SDZ PCO 400 was associated with suppression of electrical slow waves and with marked cellular hyperpolarisation. SDZ PCO 400 (0.5 and 10 microM) promoted the efflux of 86Rb+ from bovine trachealis, an effect inhibited by glibenclamide (1 microM). It is concluded that the tracheal relaxant action of SDZ PCO 400 is associated with the opening of a plasmalemmal K(+)-channel analogous to the ATP-sensitive K(+)-channel observed in insulin-secreting cells.

Tracheal relaxation induced by potassium channel opening drugs: its antagonism by adrenergic neurone blocking agents.

1. We have studied the ability of some adrenergic neurone blocking agents to inhibit the tracheal relaxant actions of isoprenaline, theophylline and the potassium channel openers (KCOs) BRL 38227, pinacidil and RP 52891. 2. BRL 38227, isoprenaline, pinacidil, RP 52891 and theophylline each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. The maximal relaxant effects of isoprenaline and pinacidil were equal to that of theophylline. In contrast, the maximal effects of BRL 38227 and RP 52891 were approximately 85-95% of that of theophylline. 3. Guanethidine (5-500 microM) did not itself modify the spontaneous tone of the trachealis muscle but antagonized BRL 38227 in a concentration-dependent manner. Guanethidine (50 microM) also antagonized pinacidil and RP 52891. However, guanethidine did not antagonize either isoprenaline or theophylline. 4. Bretylium (50 microM) did not itself modify the spontaneous tone of the trachealis muscle but antagonized BRL 38227, pinacidil and RP 52891. Bretylium did not antagonize either isoprenaline or theophylline. 5. Guanidine (50 and 500 microM) did not itself modify the spontaneous tone of the trachea and failed to modify the tracheal relaxant activity both of BRL 38227 and theophylline. 6. BRL 38227 (1 and 10 microM) stimulated, in a concentration-dependent manner, the efflux of 86Rb+ from strips of bovine trachealis muscle that had been pre-loaded with the radiotracer. Guanethidine (50 microM), bretylium (50 microM) and debrisoquine (50 microM) did not themselves modify the efflux of 86Rb+ from bovine trachealis but each of these agents markedly inhibited the stimulant effect of BRL 38227 (10 microM) on 86Rb+ efflux.7. It is concluded that the adrenergic neurone blocking agents guanethidine and bretylium can inhibit the tracheal relaxant actions of KCOs such as BRL 38227, pinacidil and RP 52891 without antagonizing isoprenaline or theophylline. The ability of the adrenergic neurone blocking agents to antagonize BRL 38227 in promoting 86Rb+ efflux from trachealis muscle may suggest that the adrenergic neurone blocking agents act to prevent the opening of the plasmalemmal K+-channel that is involved in the tracheal relaxant actions of the KCOs.

Potassium channel activators and bronchial asthma.

The cromakalim-like KCOs relax airways smooth muscle by an action that is associated with the opening of plasmalemmal K(+)-channels. The K(+)-channel involved may be analogous to the ATP-sensitive K(+)-channel identified in pancreatic beta-cells. It is unlikely to be open under normal circumstances and plays little role in determining the strong outward rectifying behaviour of the plasmalemma of the airways smooth muscle cell. K(+)-channel opening may cause relaxation of the airways smooth muscle cell by mechanisms additional to inhibition of Ca2+ influx through L-type VOCs. The cromakalim-like KCOs have bronchodilator activity in vivo and can depress NANC excitatory neuroeffector transmission in the lung at concentrations smaller than those required to relax airways smooth muscle. The mechanism of action of cromakalim in alleviating nocturnal asthma may not involve direct relaxation of airways smooth muscle. It is possible that cromakalim may instead act to inhibit the mechanisms underlying airway hyper-reactivity.

Potassium channel opening drugs and the airways.

1. Potassium channel opening drugs (KCOs) include benzopyrans such as cromakalim, cyanoguanidines such as pinacidil and tetrahydrothiopyrans such as RP 49356. 2. While clinical trials have indicated that cromakalim may be of benefit in the treatment of nocturnal asthma, it remains to be determined whether KCOs will find a place in our armamentarium of clinically useful anti-asthma agents. 3. KCOs inhibit the spontaneous tone of airways smooth muscle in vitro, an action associated with membrane hyperpolarization towards the potassium equilibrium potential and with the promotion of 86Rb+ or 42K+ efflux from the muscle cells. KCOs suppress spasm of airways smooth muscle evoked by low (< 40 mM) but not high (> 40 mM) concentrations of KCl. Their relaxant effects in airways smooth muscle can be attenuated by a variety of agents (including sulphonylureas) known to inhibit the opening of plasmalemmal K(+)-channels. 4. The KCOs open an ATP-sensitive K(+)-channel (KATP) in the plasmalemma. KATP is not open under normal circumstances and does not play an important role in determining the strong outward rectifying behavior of the cell membrane. The biochemical mechanisms by which the KCOs promote the opening of KATP remain to be elucidated but probably do not involve channel phosphorylation consequent to the intracellular accumulation of cAMP. 5. By causing hyperpolarization of the plasmalemma, the KCOs inhibit the cellular influx of Ca2+ through voltage-dependent channels. Relaxation follows both as a direct consequence of the fall in cytosolic free Ca2+ and also as a consequence of reduced production of phosphoinositide second messengers. The KCOs may also inhibit Ca2+ uptake by, and hence Ca2+ release from, the sarcoplasmic reticulum. 6. KCOs can inhibit cholinergic and non-adrenergic, non-cholinergic (NANC) excitatory neuroeffector transmission in the airways by glibenclamide-sensitive mechanisms which may involve inhibition of neurotransmitter release. The KCOs do not attenuate NANC inhibitory neuroeffector transmission, suggesting that KATP may not be expressed in neurones of this type. 7. The active enantiomer of cromakalim has been found to be effective in alleviating nocturnal asthma at plasma concentrations just threshold for relaxing human airways smooth muscle in vitro. The clinical efficacy of cromakalim may therefore depend on an action other than the direct relaxation of airways smooth muscle. Animal studies indicate that KCOs can reduce airway hyper-reactivity at sub-bronchodilator doses. The mechanism of this effect remains to be elucidated and may not crucially depend upon inhibition of neurotransmitter release within the lung.

Potassium channel opening drugs and the airways

1. Potassium channel opening drugs (KCOs) include benzopyrans such as cromakalim, cyanoguanidines such as pinacidil and tetrahydrothiopyrans such as RP 49356. 2. While clinical trials have indicated that cromakalim may be of benefit in the treatment of nocturnal asthma, it remains to be determined whether KCOs will find a place in our armamentarium of clinically useful anti-asthma agents. 3. KCOs inhibit the spontaneous tone of airways smooth muscle in vitro, an action associated with membrane hyperpolarization towards the potassium equilibrium potential and with the promotion of 86Rb+ or 42K+ efflux from the muscle cells. KCOs suppress spasm of airways smooth muscle evoked by low (< 40 mM) but not high (> 40 mM) concentrations of KCl. Their relaxant effects in airways smooth muscle can be attenuated by a variety of agents (including sulphonylureas) known to inhibit the opening of plasmalemmal K(+)-channels. 4. The KCOs open an ATP-sensitive K(+)-channel (KATP) in the plasmalemma. KATP is not open under normal circumstances and does not play an important role in determining the strong outward rectifying behavior of the cell membrane. The biochemical mechanisms by which the KCOs promote the opening of KATP remain to be elucidated but probably do not involve channel phosphorylation consequent to the intracellular accumulation of cAMP. 5. By causing hyperpolarization of the plasmalemma, the KCOs inhibit the cellular influx of Ca2+ through voltage-dependent channels. Relaxation follows both as a direct consequence of the fall in cytosolic free Ca2+ and also as a consequence of reduced production of phosphoinositide second messengers. The KCOs may also inhibit Ca2+ uptake by, and hence Ca2+ release from, the sarcoplasmic reticulum. 6. KCOs can inhibit cholinergic and non-adrenergic, non-cholinergic (NANC) excitatory neuroeffector transmission in the airways by glibenclamide-sensitive mechanisms which may involve inhibition of neurotransmitter release. The KCOs do not attenuate NANC inhibitory neuroeffector transmission, suggesting that KATP may not be expressed in neurones of this type. 7. The active enantiomer of cromakalim has been found to be effective in alleviating nocturnal asthma at plasma concentrations just threshold for relaxing human airways smooth muscle in vitro. The clinical efficacy of cromakalim may therefore depend on an action other than the direct relaxation of airways smooth muscle. Animal studies indicate that KCOs can reduce airway hyper-reactivity at sub-bronchodilator doses. The mechanism of this effect remains to be elucidated and may not crucially depend upon inhibition of neurotransmitter release within the lung

Effects of cromakalim on neurally-mediated responses of guinea-pig tracheal smooth muscle.

1. The ability of cromakalim to modulate several different types of neuroeffector transmission has been assessed in guinea-pig isolated trachea. 2. In trachea treated with propranolol (10(-6) M) and indomethacin (2.8 x 10(-6) M), stimulation of the extrinsic vagal nerves evoked contractions which were blocked by hexamethonium (5 x 10(-4) M) or by tetrodotoxin (TTX; 10(-6) M). Cromakalim (10(-5) M) caused a two fold rightward shift of the frequency-response curve. 3. In carinal trachea treated with propranolol and indomethacin, transmural stimulation evoked an initial, rapid contraction followed by a more sustained secondary contraction. The initial, rapid contractile response was virtually ablated by atropine (10(-6) M) or by TTX but was resistant to hexamethonium. Cromakalim (10(-8)-10(-5) M) caused a concentration-dependent rightward shift of the frequency-response curve for the initial contraction. 4. In carinal trachea treated with atropine, propranolol and indomethacin, transmural stimulation evoked only the secondary (non-adrenergic, non-cholinergic (NANC] contractile responses. These were markedly reduced by TTX but were resistant to hexamethonium. Cromakalim (10(-8)-10(-5) M) suppressed the NANC contractile responses in a concentration-dependent manner. This action could be offset by glibenclamide (10(-6) M). 5. In trachea treated with atropine, histamine (10(-4) M), propranolol and indomethacin, transmural stimulation evoked NANC relaxant responses. Cromakalim (up to 10(-5) M) was without effect on the frequency-response curve for the stimulation of NANC inhibitory nerves. 6. Tested on trachea bathed by drug-free Krebs solution, cromakalim (10(-7)-10(-5) M) caused concentration-dependent suppression of tracheal tone. In trachea treated with propranolol and indomethacin, cromakalim (10- 7-1O- 5 M) caused concentration-dependent antagonism of acetylcholine (ACh). In trachea treated with atropine, propranolol and indomethacin, cromakalim (up to 10- 5M) failed to antagonize effects of either histamine or substance P.7. It is concluded that cromakalim can inhibit cholinergic (excitatory) neuroeffector transmission in the trachea but only at a concentration having demonstrable inhibitory activity against the action of exogenous ACh and the spontaneous tone of the airways smooth muscle. In contrast, cromakalim may depress NANC excitatory (putative peptidergic) neuroeffector transmission at a concentration below that exerting inhibitory activity on airways smooth muscle. Cromakalim does not concurrently depress NANC inhibitory neuroeffector transmission. Depression of NANC excitatory neuroeffector transmission could explain the ability of cromakalim to suppress airway hyperreactivity or bronchial asthma at doses lacking direct relaxant effect on airways smooth muscle.