R.W. Foster

Guinea‐pig isolated trachealis: the effects of charybdotoxin on mechanical activity, membrane potential changes and the activity of plasmalemmal K+‐channels

1 A study has been made, in guinea‐pig isolated trachealis, of the effects of charybdotoxin in modulating (a) the activity of large conductance K+‐channels, (b) the spontaneous electrical activity of intact cells and (c) the mechanical effects of some bronchodilator drugs. 2 Single smooth muscle cells were isolated from guinea‐pig trachealis by enzymic digestion and were studied by the patch clamp recording technique. Recordings were made from outside‐out plasmalemmal patches when the medium bathing the external surface of the patches contained 1.2 mm Ca2+ and 6 mm K+ while that bathing the cytosolic surface contained 0.1 μm Ca2+ and 140 mm K+. Charybdotoxin (100 nm), applied to the external surface of patches held at 0 mV, abolished the unitary currents associated with the opening of large conductance K+‐channels. 3 Opened segments of guinea‐pig trachea were used for the simultaneous recording of membrane potential and tension changes. In these experiments charybdotoxin (100 nm) caused the conversion of spontaneous electrical slow waves into spike‐like action potentials. This effect was accompanied by a very small reduction in resting membrane potential. 4 Tissue bath recording showed that charybdotoxin (100 nm) increased the spontaneous mechanical tone of the tissue, antagonized (2.8 fold in each case) the relaxant actions of isoprenaline and theophylline but did not antagonize the relaxant actions of cromakalim or RP 49356. 5 It is concluded that charybdotoxin is an effective inhibitor of large conductance K+‐channels in guinea‐pig trachealis cells. The ability of charybdotoxin to convert spontaneous slow waves into spike‐like action potentials suggests that the large, charybdotoxin‐sensitive, K+‐channels play an important role in determining the strong outward rectifying behaviour of the cells. The ability of charybdotoxin to antagonize isoprenaline and theophylline, but not to antagonize cromakalim and RP 49356, suggests that opening of the large conductance, charybdotoxin‐sensitive K+‐channel is implicated in the action of the former but not the latter pair of bronchodilator drugs.

A patch‐clamp study of K+‐channel activity in bovine isolated tracheal smooth muscle cells

1 Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K+‐channels in these cells were studied by the patch‐clamp recording technique. 2 Recordings were made from inside‐out plasmalemmal patches when [K+] was symmetrically high (140 mm) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 μm. Large unitary currents of both Ca2+‐dependent and ‐independent types were observed. Measured between +20 and +40 mV, the slope conductances of the channels carrying these currents were 249 ± 18 pS and 268 ± 14 pS respectively. 3 Lowering [K+] on the cytosolic side of the patches from 140 to 6 mm, shifted the reversal potentials of the two types of unitary current from approximately zero to ≫ +40 mV, suggesting that both currents were carried by K+‐channels. 4 The Ca2+‐dependent and ‐independent K+‐channels detected in inside‐out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1–10 mm; Cs+, 10 mm; Ba2+, 1–10 mm; quinidine, 100 μm) applied to the cytosolic surface of the patches. 5 Recordings were made from outside‐out plasmalemmal patches when [K+] was symmetrically high (140 mm) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 μm. Ca2+‐dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 ± 5 pS. 6 Activity of the Ca2+‐dependent K+‐channel detected in outside‐out patches could be inhibited by application of TEA (1 mm), Cs+ (10 mm), Ba2+ (10 mm) or quinidine (100 μm) to the external surface of the patch. 4‐Aminopyridine (4‐AP; 1 mm) was ineffective as an inhibitor. 7 The activity of the Ca2+‐dependent K+‐channel recorded from outside‐out patches was reversibly inhibited by charybdotoxin (100 nm). 8 When whole‐cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2+] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nm–1 μm) applied to the external surface of the cell. 9 We conclude that bovine trachealis cells are richly endowed with charybdotoxin‐sensitive, large conductance, Ca2+‐dependent K+‐channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2+‐independent K+‐channels remains unclear.

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

1 SCA40 (1nm − 10 μm), isoprenaline (1–300 nm) and levcromakalim (100 nm − 10 μm) each produced concentration‐dependent suppression of the spontaneous tone of guinea‐pig isolated trachea. Propranolol (1 μm) markedly (approximately 150 fold) antagonized isoprenaline but did not antagonize SCA40. The tracheal relaxant action of SCA40 was unaffected by suramin (100 μm) or 8‐(p)‐sulphophenyltheophylline (8‐SPT; 140 μm). 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 μm) 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 μm) prevented the rise in tissue tone and the antagonism of SCA40 and isoprenaline induced by charybdotoxin. 4 Quinine (30 μm) 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 μm) prevented the antagonism of SCA40 induced by quinine. 5 Tested on spontaneously‐beating guinea‐pig isolated atria SCA40 (1 nm − 10 μm) increased the rate of beating in a concentration‐dependent manner. Over the concentration‐range 1 μm − 10 μm, 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 μm) 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 μm, its relaxant activity was accompanied both by slow wave suppression and by plasmalemmal hyperpolarization. 7 SCA40 (10 nm − 100 μm) 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 μm − 10 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 μm) does not involve the activation of β‐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 μm) is more likely to depend upon its selective inhibition of the type III isoenzyme of cyclic nucleotide PDE. At concentrations above 1 μm, SCA40 exerts more general inhibition of the isoenzymes of cyclic nucleotide PDE and may then promote the opening of BKCa channels.

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 (10−6m) and indomethacin (2.8 × 10−6m), stimulation of the extrinsic vagal nerves evoked contractions which were blocked by hexamethonium (5 × 10−4m) or by tetrodotoxin (TTX; 10−6m). Cromakalim (10−5m) 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 (10−6m) or by TTX but was resistant to hexamethonium. Cromakalim (10−8–10−5m) 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 (10−8–10−5m) suppressed the NANC contractile responses in a concentration‐dependent manner. This action could be offset by glibenclamide (10−6m). 5 In trachea treated with atropine, histamine (10−4m), propranolol and indomethacin, transmural stimulation evoked NANC relaxant responses. Cromakalim (up to 10−5m) 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 (10−7–10−5m) caused concentration‐dependent suppression of tracheal tone. In trachea treated with propranolol and indomethacin, cromakalim (10−7–10−5m) 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.

Mechanical, biochemical and electrophysiological studies of RP 49356 and cromakalim in guinea-pig and bovine trachealis muscle.

Experiments have been performed using guinea-pig and bovine trachealis in order to determine whether cromakalim and RP 49356 share the same relaxant action and to analyse the mechanisms underlying this action. RP 49356 was approximately 3 times less potent than cromakalim in suppressing the spontaneous tone of guinea-pig trachea and, like cromakalim, was antagonised by glibenclamide and by phentolamine. Biochemical studies showed that relaxant concentrations of cromakalim and RP 49356 did not alter the cAMP or cGMP content of guinea-pig trachealis muscle and did not inhibit cAMP or cGMP hydrolysis by tracheal homogenates. Like cromakalim, RP 49356 caused marked hyperpolarisation of guinea-pig trachealis cells. Patch clamp recording using inside-out membrane patches from bovine trachealis showed that cromakalim, RP 49356, glibenclamide and phentolamine were each without effect on the open state probability (Popen) of large conductance, Ca(2+)-activated K(+)-channels. We conclude that cromakalim and RP 49356 share a similar action in opening K(+)-channels in the trachealis cell membrane. This action probably does not involve the intracellular accumulation of cyclic nucleotides and the channel involved is not the large conductance, Ca(2+)-dependent K(+)-channel.

β-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 β‐adrenoceptor subtypes mediates the opening of plasmalemmal K+‐channels, (b) gaining further insight into the properties of the novel, long‐acting β‐adrenoceptor agonist, salmeterol and (c) clarifying the role of K+‐channel opening in mediating the relaxant actions of agonists at β‐adrenoceptors. 2 Noradrenaline (10 nm–100 μm) caused a concentration‐dependent increase in the rate of beating of guinea‐pig isolated atria. The selective β1‐adrenoceptor blocking drug, CGP 20712A (100 nm–10 μm) caused concentration‐dependent antagonism of noradrenaline. The selective β2‐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 μm), isoprenaline (1–100 nm), procaterol (0.1–30 nm), salbutamol (1 nm– 1 μm), salmeterol (1–100 nm) and theophylline (1 μm– 1 mm) each caused concentration‐dependent suppression of the spontaneous tone of guinea‐pig isolated trachealis. 4 ICI 118551 (10 nm– 1μm) antagonized isoprenaline, procaterol and salmeterol in suppressing the spontaneous tone of the isolated trachea. The antagonism was concentration‐dependent. In contrast, ICI 118551 (1 μm) antagonized neither cromakalim nor theophylline. CGP 20712A (up to 1 μm) failed to antagonize cromakalim, isoprenaline, procaterol, salmeterol or theophylline. In trachea treated with indomethacin (2.8 μm) and carbachol (10 μm), salmeterol (1 μm) 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 μm), isoprenaline (100 nm), procaterol (10 nm) and salbutamol (10nm −1 μm) were accompanied by the suppression of spontaneous electrical slow waves and by cellular hyperpolarization. In contrast, the relaxant effects of salmeterol (10 nm–1 μm) were not accompanied by significant cellular hyperpolarization. 6 CGP 20712A (1 μm) 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 μm) nor ICI 118551 (100 nm) inhibited the hyperpolarization induced by cromakalim (10 μm). Salmeterol (1 μm) inhibited the hyperpolarization induced by isoprenaline (100 nm) but not that induced by cromakalim (10 μm). 7 It is concluded that activation of either β1‐ or β2‐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 β2‐adrenoceptors as opposed to β1‐adrenoceptors nor a non‐specific action in stabilizing the cell membrane. Instead, it may reflect low intrinsic efficacy of the drug at β2‐adrenoceptors. The opening of plasmalemmal K+‐channels plays a supportive rather than a crucial role in mediating the tracheal relaxant actions of agonists at β‐adrenoceptors.

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 μm) and theophylline (1–1000 μm) 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 μm) profoundly antagonized the tracheal relaxant action of isoprenaline but not that of AH 21–132. 4 In indomethacin (2.8 μm)‐treated tissues, tone was induced by K+‐rich (120 mm) Krebs solution, acetylcholine (ACh, 1 mm) or histamine (200 μm). 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 μm), AH 21–132 (0.1–100 μm) 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. 6 In tissues treated with both indomethacin (2.8 μm) and ACh (1 mm), the removal of tracheal epithelium caused a small but significant leftward shift in the log concentration‐relaxation curve for AH 21–132 but did not alter that for theophylline. 7 In tissues treated with indomethacin (2.8 μm) and maintained at 12°C, theophylline (0.1–3.2 mm) caused concentration‐dependent spasm. This effect was not shared by AH 21–132. 8 AH 21–132 (0.1–1000 μm) more potently inhibited the activity of cyclic AMP‐dependent than of cyclic GMP‐dependent phosphodiesterase derived from homogenates of guinea‐pig trachealis. Theophylline, too, inhibited these enzymes but was less potent in each case than AH 21–132 and did not exhibit selectivity for the cyclic AMP‐dependent enzyme. 9 It is concluded that AH 21–132 exerts a non‐specific (i.e. effective no matter what agent is used to support tone) relaxant effect on the trachealis muscle which does not involve the activation of β‐adrenoceptors. The profile of the relaxant action of AH 21–132 more closely resembles that of theophylline than that of isoprenaline. However, AH 21–132 can be differentiated from theophylline in that: (a) its relaxant potency is increased by epithelial removal; (b) it does not cause tracheal spasm; (c) it exhibits selectivity as an inhibitor of cyclic AMP‐dependent as opposed to cyclic GMP‐dependent phosphodiesterase. It is possible that the relaxant effects of AH 21–132 are related to its ability to inhibit cyclic nucleotide phosphodiesterases.

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°G The relaxant response was restored on rewarming to 37°C and was abolished by indomethacin (2.8 μm). Cooling to 22°C in the presence of indomethacin revealed spasmogenic responses to caffeine which were abolished on rewarming to 37°C. 2 Trachealis treated with indomethacin (2.8 μm) was repeatedly dosed with acetylcholine (ACh, 10 μm). Caffeine (1 or 10 mm), added as each ACh‐induced spasm reached equilibrium, transiently augmented but then suppressed the spasm. On cooling from 37°C to 12°C, the increment in spasm evoked by caffeine increased relative to the spasm evoked by ACh. 3 Trachealis treated with indomethacin (2.8 μm) was repeatedly dosed with caffeine (10 mm). At 37°C caffeine had little effect but it caused spasm when the tissue was cooled to 32°C. Spasm amplitude increased as cooling progressed to 12°C. Similar results were obtained with caffeine (1 mm). 4 At 37°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 > theophylline > caffeine > theobromine > xanthine > TMX. 5 In trachealis treated with indomethacin (2.8 μm) and maintained at 12°C, the xanthines each caused concentration‐dependent spasm. The rank order of potency was theobromine ≥ theophylline ≥ caffeine ≥ enprofylline > xanthine > TMX. Forskolin was devoid of spasmogenic activity. 6 Trachealis treated with indomethacin (2.8 μm) and maintained at 12°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°C but at 37°C the KCl‐induced spasm was virtually abolished at its first trial 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 μm) and maintained at 12°C. The spasmogenic action represents the release of Ca2+ from intracellular sites of sequestration and may not depend on the intracellular accumulation of cyclic AMP. The rank order of spasmogenic potency of the xanthine derivatives differs markedly from their rank order of potency in suppressing the spontaneous tone of the trachealis observed at 37°C. Since, at 12°C, TMX is spasmogenic at concentrations identical to those causing relaxation at 37°C, it is likely that TMX penetrates the cell. The relaxant effects of TMX do not, therefore, indicate that methylxanthine‐induced relaxation is mediated by a receptor located on the external surface of the cell.

β‐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 β‐adrenoceptor subtypes mediates the opening of plasmalemmal K+‐channels, (b) gaining further insight into the properties of the novel, long‐acting β2‐adrenoceptor agonist, salmeterol and (c) clarifying the role of K+‐channel opening in mediating the mechano‐inhibitory actions of agonists at β‐adrenoceptors. 2 In bovine trachealis muscle strips precontracted with histamine (460 μm), isoprenaline (0.1 nm– 1 μm), procaterol (0.1–10 nm) and salmeterol (0.1–10 nm) each caused concentration‐dependent relaxation. 3 ICI 118551 (10 nm–1 μm) 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 μm) failed to antagonize isoprenaline, procaterol or salmeterol. 4 Salmeterol (1–10 μm) antagonized isoprenaline in relaxing strips of bovine trachea which had been precontracted with carbachol (1 μm). 5 Cromakalim (10 μm), isoprenaline (100 nm–10 μm), procaterol (10 nm–1 μm) and salbutamol (100 nm–10 μm) each promoted the efflux of 86Rb+ from strips of bovine trachealis muscle preloaded with the radiotracer. In contrast, salmeterol (100 nm–10 μm) failed to promote 86Rb+ efflux. 6 CGP 201712A (1 μm), ICI 118551 (100 nm) and salmeterol (1 μm) did not themselves modify 86Rb+ efflux from trachealis muscle strips, nor did they affect the promotion of 86Rb+ efflux induced by cromakalim (10 μm). In contrast, CGP 20712A (1 μm) and ICI 118551 (100 nm) were each able to inhibit the promotion of 86Rb+ efflux induced by isoprenaline (1 μm) or procaterol (100 nm). Furthermore, salmeterol (10 μm) inhibited isoprenaline (1 μm)‐induced promotion of 86Rb+ efflux. 7 It is concluded that, in bovine trachealis, activation of either β1‐ or β2‐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 β2‐ as opposed to β1‐adrenoceptors, but rather its low intrinsic efficacy at β2‐adrenoceptors. The opening of plasmalemmal K+‐channels plays a supportive rather than a crucial role in mediating the mechano‐inhibitory effects of agonists at β‐adrenoceptors acting on trachealis muscle.

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.