Influence of osmolarity of solutions used for K+ contraction on relaxant responses to pinacidil, verapamil, theophylline and terbutaline in isolated airway smooth muscle.

Concentration-relaxation profiles for pinacidil, verapamil, terbutaline and theophylline were studied in guinea-pig trachealis contracted by two commonly applied techniques for K+ depolarization. All drugs were much less effective on contractions induced by hyperosmolar 124 mMn K+ solution (added KCl) than on contractions elicited by an isoosmolar 124 mM K+ Krebs solution (substituted KCl). The maximal relaxant responses were (isoosmolar K+/hyperosmolar K+): pinacidil 100%/40%, verapamil 100%/60%, theophylline 100%/0%, terbutaline 50%/0%. Addition of mannitol to establish the same hyperosmolarity as with 124 mM KCl also produced contraction of guinea-pig trachealis. Concentration-relaxation curves for the drugs on mannitol-induced contractions had close resemblance to those obtained in hyperosmolar 124 mM K+ solution. When contraction was elicited by 30 mM K+, pinacidil showed seven times higher relaxant potency in hyperosmolar compared to isoosmolar solution, whereas the relaxant responses to verapamil, theophylline and terbutaline were not influenced by osmolarity. When K+ depolarization is used as a tool for evaluation of drug action in airway smooth muscle, the two different techniques produce dissimilar results. The influence of hyperosmolarity per se appears to be an important and unwanted feature when K+ depolarization is produced by addition of KCl.     

Differential relaxant responses to pinacidil of smooth muscle preparations contracted by a high concentration of potassium in isoosmolar and hyperosmolar solutions

Contractions were produced in guinea-pig trachealis, aorta and pulmonary artery by depolarization with 124 mM K+ using two commonly applied techniques. Addition of KCl to the organ bath solution making it hyperosmolar induced slowly developing contractions, which were only weakly inhibited by pinacidil. Hyperosmolar mannitol-induced contractions showed similar characteristics. In contrast, contractions elicited by isoosmolar K+ Krebs solution developed more rapidly and could be completely suppressed by pinacidil (10(-6)-10(-3) M) in a concentration-dependent manner. The findings explain previously published discrepant results on the relaxant response to pinacidil of smooth muscle preparations contracted by high concentrations of K+, and indicate other mechanisms of action for pinacidil in addition to K+ channel opening, in the concentration range 10(-6)-10(-3) M.     

Glibenclamde blocks the relaxant action of pinacidil and cromakalim in airway smooth muscle

Concentration-relaxation curves for pinacidil and cromakalim were obtained in isolated guinea-pig tracheas contracted by histamine. Pinacidil produced complete relaxation with an EC₅₀ value of 2.8 μM. The antidiabetic concentration-relaxation curve for pinacidil without changing the maximal relaxant response. Cromakalim produced 85% relaxation with an EC₅₀ value of 1.1 μM. Glibenclamide (0.1 μM) displaced the concentration-relaxation curve to the right and at higher concentrations (1–10 μM) caused nearly complete suppression of the maximal relaxant response to cromakalim. Glibenclamide not only prevented the effects of pinacidil and cromakalim but also produced a concentration-dependent and complete reversal of submaximal relaxations produced by these drugs. Glibenclamide was a selective antagonist of the relaxation of airway smooth muscle induced by pinacidil and cromakalim. Concentration-relaxation curves for theophylline, terbutaline and verapamil were unaffected by glibenclamide.     

Contraction Induced Either by Iso-osmolar or Hyper-osmolar Potassium-rich Solutions Influences Relaxant Responses to Pinacidil and Verapamil in Rat Isolated Aorta

Abstract— Relaxant responses of pinacidil and verapamil were studied in rat isolated aorta contracted by either iso-osmolar or hyper-osmolar potassium-rich solutions. Relaxant response profiles of pinacidil and verapamil in rat isolated aorta contracted by 124 mm K⁺ hyper-osmolar Krebs solutions showed marked reductions in inhibiting E_max values and substantial increases in corresponding IC50 values in comparison with results obtained at iso-osmolar conditions. Changes in the slopes of the fitted log concentration-relaxation curves were also observed, whereas pinacidil relaxation curves obtained after initial contraction induced by 30 mm K⁺ Krebs solutions were only slightly influenced by osmolarity, a definite decrease in E_max occurred for verapamil at hyper-osmolar conditions. Initial tension development was much slower and maximal tension lower when induced by 124 mm K⁺ in hyper-osmolar compared with iso-osmolar Krebs solutions. After incubation in Ca²⁺-deprived EGTA-containing Krebs solutions the maximal tension produced by 124 mm K⁺ iso-osmolar Krebs solution in rat aorta was nearly 95% reduced, whereas it was only reduced by 50% at hyper-osmolar conditions. Hyper-osmolarity as established by direct addition of potassium chloride to Krebs solutions in order to induce contraction in vascular smooth muscle could influence the in-vitro action profile, potency and intrinsic activity of the two vascular relaxant drugs.     

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 (P_open) of large conductance, Ca²⁺-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, Ca²⁺-dependent K⁺-channel.     

Relaxant Effects of the Potassium Channel Activators BRL 38227 and Pinacidil on Guinea-pig and Human Airway Smooth Muscle,and Blockade of Their Effects by Glibenclamide and BRL 31660

Summary: The airways relaxant effects and mechanism of action of the potassium channel activators BRL 38227 and pinacidil have been compared in guinea-pig and human airways. BRL 38227 was a potent relaxant in guinea-pig isolated trachealis (IC₅₀=4.9 × 10^-7 M against spontaneous tone) and human isolated bronchi (IC₅₀=4.75 × 10^-7 M against histamine-induced tone) and was eight- and six-fold more potent respectively than pinacidil. The relaxant effects of both compounds were shown to be markedly attenuated by glibenclamide (10^-5 M) and BRL 31660 (10^-5 M), with the nature of the blockade being species/tissue dependent. Glibenclamide (20 mg/kg iv) also inhibited the protective effects of BRL 38227 (50 μg/kg iv) and pinacidil (500 μg/kg iv) on histamine-induced changes in airways resistance and dynamic compliance in the anaesthetized guinea-pig, although the effects were short-lived. That both BRL 38227 and pinacidil owed their relaxant effects to potassium channel activation was supported by their ability to stimulate ^42/43 K efflux from guinea-pig trachealis preloaded with the radiotracer at concentrations of 10^-7-10^-5 M and 10^-5 M respectively. Pretreatment with either glibenclamide (10^-5 M) or BRL 31660 (10^-5 M) ablated the response to both compounds. These studies show that two mechanistically distinct potassium channel blockers, glibenclamide and BRL 31660, do not substantially differentiate between the actions of BRL 38227 and pinacidil, although differences do occur, particularly at high concentrations in vitro.     

The dissociation constant of verapamil estimated from its effect on Ca concentration-tension curves in guinea-pig tracheal muscle

Concentration-tension curves for calcium ions (Ca²⁺) were studied in indomethacin-treated guinea-pig tracheal muscle in the presence of different concentrations of carbachol in media containing 5.9 mM K⁺ or 40 mM K⁺. The effect of verapamil was investigated taking into account the steepness (the Hill coefficient) of the Ca²⁺ curve. When carbachol (1 μM) was added to 40 mM K⁺ solution, the Ca²⁺ concentration to produce half maximum tension (EC₅₀) was reduced from 0.2 mM to 0.08 mM and the Hill coefficient was increased from 1.4 to 2.0, respectively. In the presence of carbachol (1 μM), the Ca²⁺ concentration-tension curve was not much influenced by increasing the K⁺ concentration from 5.9 to 40 mM K⁺. Verapamil (0.5 μM) shifted the Ca²⁺ concentration-tension curve to the right in a parallel manner under all experimental conditions, the shift being greater with curves having a smaller Hill coefficient. The dissociation constant of verapamil was not altered by carbachol when estimated from the shift of the curve if the Hill coefficient is taken into consideration. It is concluded that the relatively low susceptibility of carbachol-induced contractions to verapamil in the presence of 40 mM K⁺, compared with these produced by K⁺ alone, is not due to a decreased verapamil affinity but to improved Ca²⁺-response coupling.     

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.     

Effects of Antidiabetic Sulphonylureas,Cromakalim and their Interaction in Guinea-pig Isolated Tracheal Smooth Muscle

Summary: Glibenclamide, glipizide and glibornuride showed dual effects in guinea-pig isolated trachea. The drugs antagonized the relaxant response to the K⁺ channel opener cromakalim (order of effectiveness: glibenclamide > glipizide > glibornuride) and at concentrations of 1-1000 μM produced airway smooth muscle relaxation (order of potency: glibenclamide > glipizide = glibornuride). Gliclazide, tolbutamide and chlorpropamide did not antagonize cromakalim nor did the two latter drugs produce tracheal relaxation. The sulphonylureas and cromakalim were compared as airway relaxants against a panel of different spasmogens. The order of tissue responsiveness for the sulphonylureas was: spontaneous tone = LTD₄ > PGF_2α = histamine = 30 mM K⁺ > carbachol and for cromakalim: spontaneous tone = LTD₄ = PGF_2α = histamine > carbachol > 30 mM K⁺. Glibenclamide, but not cromakalim, relaxed contractions induced by 124 mM K⁺. Phentolamine and Ba²⁺, which are reported blockers of ATP-regulated K⁺ channels, failed to influence sulphonylurea-induced airway smooth muscle relaxation. Glibenclamide reversed tracheal relaxation produced by cromakalim, whereas cromakalim failed to reverse relaxation induced by glibenclamide. The mechanism for the additional property of sulphonylureas to relax airway smooth muscle is unclear, but the results do not support a role for involvement of cromakalim-sensitive K⁺ channels.     

Influence of depolarization on vasorelaxant potency and efficacy of Ca2+ entry blockers, K+ channel openers, nitrate derivatives, salbutamol and papaverine in rat aortic rings

We examined the effects of various KCl concentrations on the actions of some vasodilators belonging to different pharmacological classes in rat aortic rings. In some experiments, tissues were precontracted with noradrenaline after blocking voltage-dependent channels to assess the effects of depolarisation unaccompanied by the entry of extracellular Ca²⁺ into the cytosol. Concentration/response curves for the vasorelaxant effect of calcium entry blockers (e.g. diltiazem), K⁺ channel openers (e.g. aprikalim), nitrate derivatives (e.g. nitroglycerin), a β₂-adrenergic agonist (salbutamol) and papaverine were obtained by using endothelium-denuded rat aortic rings precontracted with KCl (20–60 mM) to determine the potencies and efficacies of the drugs. The efficacies and potencies of calcium entry inhibitors were virtually independent of the [KCl]. A reduction in the potency (up to 18-fold) of papaverine occurred without changes in efficacy when the [KCl] was raised from 20 to 60 mM. The decline in potency was even greater for nitrate-like compounds. The potency of K⁺ channel openers in aortic rings precontracted with 30 mM KCl decreased by three- to sixfold compared with those precontracted with 20 mM KCl. With the exception of pinacidil, the efficacy of these agents already started to decline in preparations precontracted with 25 mM KCl and was virtually zero in preparations precontracted with 60 mM KCl. In contrast to other K⁺ channel openers, the vasorelaxant action of pinacidil was relatively resistant to glibenclamide. Salbutamol produced only a slight relaxation even in preparations precontracted with 20 mM KCl. In nitrendipine-pretreated, noradrenaline-precontracted aortic rings, the vasorelaxant effects of aprikalim, but not those of linsidomine or papaverine, declined when the [KCl] of the bathing medium was increased. In conclusion, the vasorelaxant potency and efficacy of calcium entry blockers is independent of the [KCl] used to precontract rat aortic rings, and thus, of the degree of membrane depolarisation. In contrast, increasing the [KCl] strongly reduces the potency and the efficacy of K⁺ channel openers not only in this preparation but also in noradrenaline-precontracted rings in which the entry of extracellular Ca²⁺ was prevented with nitrendipine. This indicates that, with the exception of pinacidil, the vasorelaxant activity of K⁺ channel openers depends on the degree of membrane depolarisation. Finally, the vasorelaxant potency and efficacy of nitrate-like compounds and papaverine are independent of depolarisation per se but they are markedly affected by the influx of Ca²⁺ accompanying elevated [KCl]. Thus, the degree of vessel depolarisation should be taken into consideration when attempting to compare potencies and efficacies among vasorelaxant agents. Received: 4 May 1998 / Accepted: 6 July 1998     

Comparison of the Effects of a New Vasodilator Pinacidil and Nifedipine on Isolated Blood Vessels

Abstract: In isolated human crural veins studied in vitro pinacidil (0.038–380 μM) caused a concentration-related inhibition of noradrenaline, 18 μM (NA) and 127 mM K⁺-induced contractions. Pinacidil was more potent in inhibiting the NA-contraction than that induced by K⁺, whereas the reverse was seen for nifedipine. At the highest concentrations greater inhibitions of the NA-induced contractions could be obtained with pinacidil than with nifedipine. The inhibitory effect of pinacidil on the K⁺-induced contractions was eliminated during a 1 hr wash-out period. In contrast to this, the inhibitory effect of nifedipine could not be eliminated during 4 hrs repeated wash-out. Pinacidil was completely devoid of inhibitory effect on ⁴⁵Ca net influx in rat aorta, whereas nifedipine caused a significant reduction of influx. The results indicate that both pinacidil and nifedipine are effective vasodilatators in human vessels. Pinacidil seems to be more effective in NA-induced contractions than does nifedipine. The mechanism of action of pinacidil cannot be attributed to an inhibitory effect on cellular calcium entry.     

Effect of Cromakalim on KCl-, Noradrenaline- and Angiotensin II-induced Contractions in the Rat Pulmonary Artery

Summary: The effect of cromakalim on vascular reactivity was studied in rat isolated pulmonary arterial strips. Cromakalim (0.1-1 μM) inhibited contractions induced by low (20-30 mM) KCl concentrations in a concentration-dependent manner. It had no effect on those elicited by 60-100 mM KCl. However, a higher concentration of cromakalim (10 μM) slightly decreased (-5 to -10%) KCl efficacy. Contractions induced by noradrenaline (NA, 0.01-1 μM) and angiotensin II (AII, 0.5-50 nM) were reduced by cromakalim (0.1-10 μM). The maximal response to NA and AII was decreased by 54 ± 6.4% and 70 ± 5.8% (n = 5), respectively, in the presence of 10 μM cromakalim. The inhibitory effect of cromakalim was not dependent on the presence of vascular endothelium. After blockade of calcium influx by verapamil (10 μM), cromakalim had no further effect on NA- and AII-induced contractions. Cromakalim (0.1-1 μM) had no effect on the amplitude of the transient contraction evoked by NA and AII in Ca²⁺-free solution. The inhibitory effect of cromakalim (1 μM) was reversed by glibenclamide (1-10 μM) and phentolamine (5-100 μM) which, however, did not alter the relaxant effect of verapamil (1 μM), papaverine (1 μM) or theophylline (1 mM). Contractions induced by NA and AII in the presence of tetraethylammonium (TEA, 10 mM) were also depressed by cromakalim. These results show that cromakalim is a potent anticonstrictor agent in the pulmonary circulation. As in other smooth muscles, its mechanism of action involves an interaction with potassium channels at the vascular smooth muscle cell membrane level.     

Calcium channels at the adrenergic neuroeffector junction in the rabbit ear artery

Summary Neurotransmitter release is dependent on influx of Ca²⁺ through voltage-operated calcium channels (VOCCs). These channels may be divided into L, N, T and P subtypes. To investigate the subtypes of VOCC involved in transmitter release from adrenergic nerves in the isolated rabbit ear artery, the effects of some subtype selective VOCC antagonists were examined on contractile responses induced by electrical field stimulation (EFS), and exposure to an isosmolar (low Na⁺, normal Cl^– content) or a hyperosmolar (normal Na^]+, high Cl^– or 60 mM K+ solution). Tetrodotoxin (TTX) and the L channel blocker nimodipine were present in the latter experiments to inhibit sodium-dependent action potential discharge and the direct contractile effect of K⁺ depolarization on the smooth muscle cells. Prazosin abolished the contractile effect of EFS, indicating that the response was elicited by activation of adrenergic nerves. The EFS-induced contractions were concentration-dependently inhibited by the N channel blocker -conotoxin (PIC₅₀ = 9.0) and the proposed L channel blocker T-cadinol (pIC₅₀ = 4.5), while nimodipine and the T channel blocker tetramethrin had no effect. The isosmolar and hyperosmolar K⁺ solutions induced a prazosin-sensitive contraction, amounting to 46% and 10% of the response to 10^–5 M noradrenaline (NA), respectively. -Conotoxin inhibited the contractile response to the hyperosmolar K⁺ solution, but not that to the isosmolar K⁺ solution. T-cadinol preferentially inhibited the response to the hyperosmolar K⁺ solution. Tetramethrin had no effect on contractions induced by either type of K⁺ solution. The contractile response to exogenous NA was unaffected by -conotoxin and tetramethrin, whereas the response was partially inhibited by both nimodipine and T-cadinol. These results suggest that NA release from adrenergic nerves in the rabbit ear artery, depend on Ca²⁺ influx through VOCCs of the N type, whereas L and T channels seem to be of minor importance. Calcium influx into the nerve terminals via a tentative Na⁺/Ca²⁺ exchange mechanism may explain the failure of -conotoxin to inhibit the adrenergic response to the isosmolar K⁺ solution.Correspondence to P. Zygmunt at the above address     

Effects of pinacidil on guinea-pig airway smooth muscle contracted by asthma mediators

Pinacidil is a new antihypertensive, direct vasodilator drug which has been classified as a K+ channel opener. The present study demonstrated a concentration-dependent relaxant activity of pinacidil in guinea-pig tracheal preparations. The potency and efficacy of pinacidil depended on the agent used to induce tracheal tone. Tracheal preparations with spontaneous tone or precontracted by different asthma mediators were completely relaxed by pinacidil. A high potency was found in spontaneously contracted preparations (EC50 = 7.8 x 10(-7) M). The EC50 values ranged from 2.3 to 5.4 x 10(-6) M in histamine-, PGF2 alpha- or LTC4-contracted preparations. When tone was induced by carbachol, the EC50 was 2.1 x 10(-5) M. In contrast, pinacidil produced incomplete relaxation and had a low potency in preparations contracted by 30 or 124 mM K+ Krebs solutions. This effect profile differed from that seen with beta 2-receptor agonists, xanthines and Ca2+ antagonists in guinea-pig trachealis and seems compatible with K+ channel opening as a primary mode of relaxation for pinacidil in airway smooth muscle.     

Differential Vasorelaxant Effects of K+-Channel Openers and Ca2+-Channel Blockers on Canine Isolated Arteries

Abstract— The vasorelaxant effects of the K⁺-channel openers, pinacidil and cromakalim, were compared with those of the Ca²⁺-channel blockers, verapamil and KB-2796 (1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)piperazine dihydrochloride), in canine isolated coronary, renal, basilar and mesenteric arteries precontracted with U46619, a thromboxane A₂ mimetic. The relaxation induced by pinacidil and cromakalim was greater in coronary than in other arteries, the magnitude of relaxation being in the order of coronary > renal > basilar > mesenteric arteries. The relaxant responses to both drugs were inhibited by glibenclamide, a blocker of ATP-sensitive K⁺ channels. The relaxation induced by verapamil and KB-2796, in contrast, was greater in basilar than in other arteries, the magnitude of relaxation being in the order of basilar > coronary > renal and mesenteric arteries. In fura-2-loaded, U46619-stimulated arteries, pinacidil and cromakalim produced a greater reduction in intracellular Ca²⁺ concentration and muscle tension in coronary than in mesenteric arteries, while verapamil and KB-2796 reduced these values more potently in basilar than in mesenteric arteries. These results suggest that K⁺-channel openers exhibit a vasorelaxant selectivity for coronary arteries, whereas Ca²⁺-channel blockers exhibit such selectivity for cerebral arteries. The selective vasorelaxant action induced by these drugs appears to correspond, in part, to their effects on the concentration of intracellular Ca²⁺.     

Effects of pinacidil on cerebral and mesenteric arteries —influence of the endothelium

Summary The effect of pinacidil on the contractile response to stepwise increases of the extracellular K⁺ concentration ([K⁺]₀) was investigated in isolated segments of human pial and mesenteric arteries and rabbit basilar and mesenteric arteries. The [K⁺]_O eliciting half maximum contraction (EC₅₀) was lower in human pial (18 mM) and rabbit basilar (27 mM) arteries than in human (33 mM) and rabbit (32 mM) mesenteric arteries, respectively. The -adrenoceptor blocker, prazosin, increased the EC₅₀ value for K⁺ from 27 to 40 mM and reduced the maximum response in rabbit mesenteric arteries, but had no effect on the K⁺-induced contraction in rabbit basilar arteries, indicating a substantial noradrenergic component of the K⁺ response in the former arteries. Removal of the endothelium decreased the EC₅₀ value for K⁺ from 27 to 15 mM in rabbit basilar arteries, whereas the K⁺ sensitivity was unaffected in rabbit mesenteric arteries. Pinacidil shifted the K⁺ concentration-response curve to the right in human and rabbit cerebral and mesenteric arteries. In rabbit basilar arteries, but not in mesenteric arteries, the shift was larger in the absence than in the presence of an intact endothelium. When endothelium-denuded rabbit arteries were compared, the inhibitory effect of pinacidil was larger in basilar than in mesenteric arteries. Thus, pinacidil inhibits K⁺-induced contractions in both cerebral and mesenteric arteries, but appears to act preferentially on endothelium-denuded rabbit basilar arteries. Provided that endothelial damage and depolarization-induced vasoconstriction are of pathophysiological importance in cerebrovascular disorders such as stroke and cerebral ischemia secondary to vasospasm after subarachnoid hemorrhage, pinacidil may have a therapeutic potential. Correspondence to E. D. Hogestatt at the above address     

Effect of potassium channel opener pinacidil on the contractions elicited electrically or by noradrenaline in the human radial artery

In order to discover an agent that can prevent spasm of the human radial artery, the aim of our study was to evaluate the effect of the K⁺ channel opener, pinacidil, on contractions in the radial artery. Contractions of the radial artery were evoked by exogenously applied noradrenaline or by electrical field stimulation (EFS, 20 Hz, neurogenic). Pinacidil induced concentration-dependent inhibition of both EFS- and noradrenaline-evoked contractions of the radial artery. Glibenclamide, a selective blocker of ATP-sensitive K⁺ channels (Kir6.x containing subunit) antagonized in the same manner the pinacidil-induced inhibition of neurogenic contractions and contractions evoked by exogenous noradrenaline. The inhibition of pinacidil relaxation by tetraethylammonium (TEA), a blocker of Ca-sensitive K⁺ (K_Ca) channels, was more pronounced in EFS-contracted preparations. A blocker of voltage-sensitive K⁺ (K_V) channels, 4-aminopyridine (4-AP), inhibited pinacidil relaxation only in EFS-contracted preparations. In order to test the presence of different K⁺ channels, immunohistochemistry of K⁺ channels expression in the radial artery was performed. The vascular wall of the human radial artery showed variable positivity with the following applied antibodies: Kv1.2, Kv1.3, Kir6.1, and K_Ca1.1. The antibodies against Kv1.6, Kv2.1, and Kir6.2 channel subunits were completely negative. These results suggest that the inhibitory effect of pinacidil on contractions of the human radial artery might be postsynaptic and associated with opening of smooth muscle Kir6.1-containing K_ATP channels. TEA- and 4-AP-sensitive K⁺ channels may also contribute to pinacidil effect in the human radial artery.     

Effects of hydralazine and verapamil on phosphorylase activity and guanosine cyclic 3′,5′-monophosphate levels in guinea-pig taenia coli

1 The roles of guanosine cyclic 3',5'-monophosphate (cyclic GMP) and calcium in the relaxation produced by hydralazine and verapamil in potassium-depolarized guinea-pig taenia coli have been investigated.2 Depolarization of isolated strips of guinea-pig taenia coli by 124 mM KCl caused sustained contractures and increases in tissue levels of cyclic GMP.3 The KCl-induced increases in cyclic GMP levels appeared to be calcium dependent. No increases in cyclic GMP levels were seen in strips of taenia coli depolarized in the absence of calcium. Readdition of calcium to the depolarizing solution contracted the muscles and increased cyclic GMP levels. When calcium was removed from the depolarizing solution during the sustained, tonic phase of a KCl-induced contracture, both tension and cyclic GMP levels returned to control values.4 Administration of 50 muM verapamil to KCl-contracted muscles completely relaxed the muscles and caused cyclic GMP levels to return to control values within 14 min. Hydralazine, 2 mM, on the other hand, relaxed the depolarized muscles without lowering cyclic GMP levels. No significant changes in cyclic AMP levels were seen in any of these experiments.5 Similar results were obtained in an analogous series of experiments in which glycogen phosphorylase activity was measured instead of cyclic GMP levels. Activation of phosphorylase during contractions of guinea-pig taenia coli had previously been reported to be a calcium-dependent phenomenon.6 It was concluded that increases in tissue levels of cyclic GMP (or of cyclic AMP) were not responsible for the relaxant effects of hydralazine or verapamil in these experiments. It was also suggested, based on our results, that verapamil exerted its relaxant effects in the depolarized taenia coli by lowering cytoplasmic calcium levels, whereas hydralazine relaxed the depolarized muscles without lowering intracellular calcium activity.     

DRUG INTERACTIONS IN CAT ISOLATED TRACHEAL SMOOTH MUSCLE

1. The interactions between some drugs that contract and relax airways smooth muscle have been investigated in the cat isolated trachea. 2. Isoprenaline and theophylline inhibited serotonin-elicited contractions more than acetylcholine-mediated responses. This was observed both in terms of the degree of inhibition and the concentration of the relaxant drug producing this inhibition. 3. The acetylcholine- and serotonin-induced contractions were inhibited more by theophylline than by isoprenaline. Isoprenaline displaced acetylcholine and serotonin response curves to the right whereas theophylline caused a flattening of the curves. 4. Isoprenaline was more effective in inhibiting serotonin contractions than acetylcholine contractions when the tracheas were bathed in K⁺ depolarizing solution, suggesting that the difference in the susceptibility of serotonin and acetylcholine contractions to isoprenaline was not dependent on the electrical membrane potential. 5. Isoprenaline inhibited the tonic component of acetylcholine contractions more than the phasic component. 6. The differences in the pharmacological responses to the contractile and relaxant drugs in cat tracheal preparations provide further examples in smooth muscle of different mechanisms by which acetylcholine and serotonin induce contraction and isoprenaline and theophylline relaxation.     

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

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