The relaxant action of nicorandil in bovine tracheal smooth muscle

The relaxant mechanisms of nicorandil were examined by comparing its effects with those of sodium nitroprusside and cromakalim in bovine tracheal smooth muscle. In preparations contracted with methacholine (0.3 μ mol/l) or high K(+)(40 mmol/l), nicorandil and sodium nitroprusside caused concentration-dependent relaxations. Their relaxant effects on high K(+) -contracted preparations were smaller than those on methacholine-contracted muscle. Cromakalim relaxed methacholine-contracted preparations, whereas it had no effect on high K(+) -contracted muscle. The inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 mol/l) completely prevented the relaxation induced by lower concentrations ( <30 μ mol/l) of nicorandil,whereas it partially attenuated relaxation caused by higher concentrations. The ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide only partially attenuated the relaxant responses to nicorandil (at 100 and 300 μ mol/l). Combination treatment with ODQ and glibenclamide almost completely prevented nicorandil-induced relaxations. The large-conductance Ca2(+) -activated K(+) channel (Maxi K(+) channel) inhibitor iberiotoxin significantly prevented the relaxations induced by lower concentrations (3 and 10 μ mol/l) of nicorandil. The preventive effect of iberiotoxin was markedly enhanced under the blockade of K(ATP) channels with glibenclamide. These results suggest that nicorandil relaxes bovine tracheal smooth muscle through 2 mechanisms: opening of K(ATP) channels and activation of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Nicorandil may also activate Maxi K(+) channels, possibly through the NO-cGMP pathway, and the interaction of K ATP channels and Maxi K(+) channels may affect the relaxant effect of nicorandilin bovine tracheal smooth muscle.     

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides in guinea pig tracheal smooth muscle

Relaxation and modulation of cyclic AMP production in response to atrial natriuretic peptides were investigated in epithelium-denuded guinea pig tracheal rings, treated with indomethacin (5 μM) and phosphoramidon (1 μM) and contracted with histamine (3 μM). Atrial natriuretic peptide (ANP) was a more potent relaxant than C-type natriuretic peptide whereas ANP-(4–23) was inactive suggesting the involvement of ANP_A receptors in the relaxant effect of ANP. ODQ (1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one, 10 μM), a selective inhibitor of soluble guanylyl cyclase, markedly inhibited the relaxant response to sodium nitroprusside. The relaxant response to ANP was not altered by ODQ demonstrating the involvement of particulate guanylyl cyclase. ANP-induced relaxations, as well as sodium nitroprusside-induced relaxations, were similarly potentiated by rolipram (4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one, 3 μM), a type IV phosphodiesterase inhibitor, and by zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one, 10 μM), a type V phosphodiesterase inhibitor. ANP-mediated response was unaffected by glibenclamide (10 μM), a selective blocker of ATP-sensitive K⁺ channels, and by apamin (1 μM), a selective blocker of small-conductance Ca²⁺-activated K⁺ channels. Iberiotoxin (100 nM) extensively prevented the relaxant effect of ANP suggesting the activation of large-conductance Ca²⁺-activated K⁺ channels. In addition, ANP (10 nM) and ANP-(4–23) (100 nM) significantly reduced forskolin (1 μM)-stimulated cAMP accumulation suggesting, for the first time, the presence of functional ANP_C receptors in guinea pig airway smooth muscle. However, relaxations to forskolin and to isoproterenol were not altered in the presence of ANP-(4–23) or ANP demonstrating that the inhibitory effect of ANP-(4–23) and ANP on adenylyl cyclase was not sufficient to alter the functional response induced by these two activators of adenylyl cyclase.     

Potentiation of beta-adrenoceptor function in bovine tracheal smooth muscle by inhibition of protein kinase C

To examine the role of contractile agonist-induced activation of protein kinase C (PKC) in functional antagonism of airway smooth muscle contraction by beta-adrenoceptor agonists, we examined the effects of the specific PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide) on isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by various concentrations of methacholine and histamine. In the absence of GF 109203X, the potency of isoprenaline (pD(2)) was gradually reduced at increasing methacholine- and histamine-induced smooth muscle tones, but the maximal relaxation (E(max)) was decreased only at higher concentrations of methacholine. In the presence of GF 109203X, pD(2) values were significantly increased for both methacholine- and histamine-induced contractions. Moreover, isoprenaline E(max) values in the presence of high concentrations of methacholine were also increased. Although both methacholine- and histamine-induced contractions were slightly reduced by GF 109203X, the changes in isoprenaline pD(2) could only partially be explained by reduced contractile tone. In contrast to isoprenaline, forskolin-induced relaxations were not affected by GF 109203X. The results indicate that PKC activation contributes to the reduced beta-adrenergic responsiveness induced by methacholine and histamine, which may involve uncoupling of the beta-adrenoceptor from the effector system. Since many mediators and neurotransmitters in allergic airway inflammation can activate PKC, this cross talk may be important in the reduced bronchodilator response of patients with severe asthma.     

Lidocaine potentiates atrial natriuretic peptide-induced relaxation of bovine tracheal smooth muscle

The effect of lidocaine on the changes in tension and guanosine 3',5'-cyclic monophosphate (cGMP) content induced by atrial natriuretic peptide (ANP) and nitric oxide (NO) was examined in bovine tracheal smooth muscle preparations contracted with methacholine (0.3 microM). Lidocaine (10 microM) did not affect the methacholine-induced tensions, whereas 100 microM lidocaine significantly (P<0.01) attenuated methacholine-induced ones. Treatment of the tracheal preparations with lidocaine (10 and 100 microM) significantly (P<0.05) augmented the relaxant responses to ANP, whereas the same procedure did not alter the responses to sodium nitroprusside, (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) or 8-bromo-cGMP. Lidocaine (100 microM) enhanced cGMP accumulation induced by ANP (0.1 microM) but not by sodium nitroprusside (0.3 microM). In contrast, mexiletine (100 microM), another class Ib antiarrhythmic, did not affect ANP- and sodium nitroprusside-induced relaxations. These results suggest that lidocaine augments ANP-induced relaxation and cGMP accumulation, probably by modulating activation mechanism of particulate guanylyl cyclase.     

Cyclic nucleotide-dependent phosphodiesterases (PDEI) inhibition by muscarinic antagonists in bovine tracheal smooth muscle

In bovine tracheal smooth muscle (TSM) strips, muscarinic antagonists (atropine, 4-DAMP, AFDX-116 and methoctramine) were able to increase simultaneously and a similar fashion the intracellular levels of cyclic nucleotides, with a cAMP/cGMP ratio higher than 2.0. These original pharmacological responses were time-and dose-dependent, exhibiting maximal values at 15 min, with a pEC(50) of 7.4 +/- 0.2 for atropine and 4-DAMP. These effects on cAMP and cGMP levels were similar to the ones obtained with isobutyl-methylxantine (IBMX, 10 microM), a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor, suggesting the involvement of PDEs in these muscarinic antagonist responses. Neither, rolipram (10 microM), a specific PDEIV inhibitor, nor zaprinast (10 microM), a PDEV inhibitor, exhibited this "atropine-like" responses. Instead, atropine enhanced the increments of cAMP levels induced by rolipram and cGMP levels by zaprinast. However, vinpocetine (20 microM), a non-calmodulin dependent PDEIC inhibitor was able to mimic these muscarinic antagonist responses in intact smooth muscle strips. In addition, in cell free systems, muscarinic antagonists inhibited the membrane-bound PDEIC activity whereas soluble (cytosol) PDEIC activity was not affected by these muscarinic drugs. These results indicate that muscarinic antagonists acting possibly as inverse agonists on M(2)/M(3)mAChRs anchored to sarcolemma membranes can initiate a new signal transducing cascade leading to the PDEIC inhibition, which produced a simultaneous rise in both cAMP and cGMP intracellular levels in tracheal smooth muscle.     

Role of K+ channels in N-acetylprocainamide-induced relaxation of bovine tracheal smooth muscle

We examined the relaxant effects of N-acetylprocainamide, the major hepatic metabolite of procainamide, on bovine tracheal smooth muscle, focusing on the possible involvement of K+ channels. N-acetylprocainamide produced a concentration-dependent and full inhibition of the tension development elicited by methacholine (0.3 or 1 microM). The potency of N-acetylprocainamide in diminishing methacholine-elicited tension development was one-half of that of procainamide. By comparison, N-acetylprocainamide inhibited high-K+ (40 mM)-induced contraction more potently than procainamide though both inhibitions were largely reduced when compared to those against methacholine-induced contraction. Iberiotoxin (30 nM), Ba(2+) (1 mM) or a combination of both agents significantly attenuated the relaxant effect of N-acetylprocainamide on methacholine-induced contraction, whereas apamin (100 nM), 4-aminopyridine (300 microM), and glibenclamide (10 microM) did not affect it. These results suggest that N-acetylprocainamide, similar to procainamide, elicits tracheal smooth muscle relaxation mainly through the activation of plasma membrane K+ channels.     

Involvement of K(+) channel in procainamide-induced relaxation of bovine tracheal smooth muscle

The relaxant effect of procainamide, a class Ia antiarrhythmic agent, was examined in bovine tracheal smooth muscle. Procainamide produced concentration-dependent decreases in tension and full relaxation in the preparations contracted with methacholine (0.3 microM). By comparison, in preparations contracted with 40 mM K(+), procainamide had only slight relaxant effects. The relaxant effects of cromakalim and salbutamol on 40 mM K(+)-contracted preparations were significantly (P<0.01) smaller than those on 0.3 microM methacholine-contracted ones. On the other hand, the concentration-response relationships for quinidine, lidocaine, mexiletine and propafenone were not so dramatically different between 0.3 microM methacholine- and 40 mM K(+)-contracted preparations. Tetraethylammonium (300 microM), iberiotoxin (30 nM) and Ba(2+) (1 mM) significantly (P<0.05) attenuated the relaxant effects of procainamide on methacholine-induced contractions, whereas apamin (100 nM), 4-aminopyridine (300 microM), and glibenclamide (10 microM) did not affect them. The inhibitory effect of a combination of iberiotoxin and Ba(2+) was greater than that of iberiotoxin or Ba(2+) alone (P<0.01). These results suggest that the activation of at least two types of K(+) (maxi-K(+) and inward rectifier K(+)) channels contributes to the procainamide-induced relaxation of bovine tracheal smooth muscle.     

Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase

Cyclic nucleotide analogs were used to study relaxation of pig coronary arteries and guinea pig tracheal smooth muscle in an attempt to determine the roles of cAMP- and cGMP-dependent protein kinases (cA-K and cG-K). In pig coronary artery strips, cGMP analogs were generally more effective than cAMP analogs in promoting relaxation of K+-induced contractions. Significant relaxation of this tissue was caused primarily by those cyclic nucleotide analogs that had high affinities for purified cG-K but not for cA-K. The low potencies of cA-K-specific analogs, as compared with cG-K-specific analogs, could not be readily explained by either unusually high susceptibilities to phosphodiesterases or low partition coefficients. The most potent cGMP analog, 8-(4-chlorophenylthio)-cGMP, exhibited a very slow reversibility of its relaxant effects in the intact tissue, consistent with its strong resistance to hydrolysis by phosphodiesterases measured in vitro. Pig coronaries contained atypically high levels of cGMP and cG-K, implying a potentially important role of this enzyme in smooth muscle function. Carbamylcholine-induced contractions of guinea pig tracheal segments were more sensitive than K+-induced pig coronary artery contractions to relaxation by cyclic nucleotide analogs. Consequently, the number of analogs that could be studied was significantly expanded. The cGMP analogs were again generally more potent, and the effectiveness of both cGMP and cAMP analogs in relaxing this preparation correlated with the Ka of the analogs for in vitro activation of cG-K, but not cA-K. A particularly strong correlation was observed when the effects of analogs modified only at the C-8 position were examined. A known target enzyme of cA-K, phosphorylase, was not activated by cG-K-specific analogs but was activated by high concentrations of the cA-K-specific analogs. Studies using cyclic nucleotide analogs support a role for cG-K, but not for cA-K, in decreasing smooth muscle tone.     

Lidocaine potentiates the relaxant effects of cAMP-elevating agents in bovine tracheal smooth muscle

The effect of lidocaine on the relaxation and accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) induced by salbutamol, forskolin and 3-isobutyl-1-methylxanthine (IBMX) was examined in bovine tracheal smooth muscle preparations precontracted with methacholine (0.3 microM). Lidocaine attenuated the methacholine-induced contraction in a concentration-dependent manner. Pretreatment of the preparations with lidocaine (100 microM) caused significant leftwards shifts of concentration/response curves for the relaxant responses to salbutamol, forskolin, and IBMX, whereas it did not change the responses to diltiazem. Similar leftwards shifts were observed when the preparations were treated with procaine (6 microM) or bupivacaine (40 microM). Lidocaine (100 microM) augmented cAMP accumulation induced by salbutamol (10 nM) and forskolin (1 microM). These results suggest that lidocaine augments the relaxant responses to cAMP-elevating agents through enhancement of cAMP accumulation.     

Beta-adrenoceptor stimulation inhibits histamine-stimulated inositol phospholipid hydrolysis in bovine tracheal smooth muscle.

1. Histamine and carbachol produced concentration-related increases in the accumulation of 3H-inositol phosphates in slices of bovine tracheal smooth muscle. 2. Noradrenaline alone produced a small stimulation of 3H-inositol phosphate accumulation which was inhibited by the alpha-adrenoceptor antagonist phentolamine. In contrast, when noradrenaline (0.1 mM) was added simultaneously with histamine it significantly reduced the inositol phosphate response to high (greater than or equal to 0.1 mM) concentrations of histamine. However, noradrenaline had no inhibitory effect on the carbachol-induced inositol phosphate response. 3. The non-selective beta-agonist isoprenaline (IC50 = 0.08 microM) and the beta 2-selective agonist salbutamol (IC50 = 0.29 microM) both produced a dose-related inhibition of the inositol phosphate response to 0.1 mM histamine. The inhibitory effect of salbutamol was antagonized by propranolol (KA = 2.4 x 10(9) M-1) and the beta 2-selective adrenoceptor antagonist ICI 118551 (KA = 1.7 x 10(9) M-1). 4. The accumulation of 3H-inositol phosphates induced by histamine increased steadily over a 40 min period after an initial lag period of 3-4 min. Following the simultaneous addition of histamine and salbutamol there was a further delay of 3-4 min before the appearance of the inhibitory effect of salbutamol. 5. The effect of histamine on inositol phosphate accumulation was accompanied by a stimulation of [3H]-inositol incorporation into membrane phospholipids which was reduced by the presence of salbutamol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of serum-induced proliferation of bovine tracheal smooth muscle cells in culture by heparin and related glycosaminoglycans.

1. The effect of heparin and related glycosaminoglycans on bovine airway smooth muscle proliferation has been investigated. 2. Foetal bovine serum stimulated division of bovine trachealis smooth muscle cells in a concentration-dependent fashion at concentrations between 1 and 30%. 3. Heparin (0.1-100 micrograms ml-1), heparan sulphate (0.1-100 micrograms ml-1) and fragmin (0.1-100 micrograms ml-1) inhibited smooth muscle division in a concentration-dependent fashion between 0.1-100 micrograms ml-1. A heparin disaccharide did not exhibit inhibition of division at 100 micrograms ml-1. 4. Dextran sulphate at molecular weights of 5 x 10(3) and 5 x 10(5) concentration-dependently inhibited division between 0.1-100 micrograms ml-1. Dextran without sulphation did not exhibit inhibition of division at 100 micrograms ml-1. 5. The magnitude of inhibition of proliferation did not reach 100% for any compounds examined at concentrations up to 100 micrograms ml-1 during incubations for 5 and 14 days. IC50 values for inhibition of proliferation ranged between 1-5 micrograms ml-1. 6. These findings suggest that heparin and related glycosaminoglycans inhibit bovine airway smooth muscle cell division.     

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

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

Functional characterization of serum- and growth factor-induced phenotypic changes in intact bovine tracheal smooth muscle

1. The present study aims to investigate whether phenotypic changes, reported to occur in cultured isolated airway smooth muscle (ASM) cells, are of relevance to intact ASM. Moreover, we aimed to gain insight into the signalling pathways involved. 2. Culturing of bovine tracheal smooth muscle (BTSM) strips for up to 8 days in the presence of 10% foetal bovine serum caused a time-dependent (t(1/2)=2.8 days) decrease in maximal contraction (E(max)) to methacholine compared to serum-deprived controls (E(max)=74+/-4% at day 8). A reduced E(max) was also found using insulin-like growth factor-1 (30 ng ml(-1)) and platelet-derived growth factor (30 ng ml(-1)), but not using epidermal growth factor (10 ng ml(-1)) (E(max)=83+/-3, 67+/-8, 100+/-4%, respectively). Similar serum and growth factor-induced changes in E(max) were found for KCl-induced contraction (65+/-9, 80+/-7, 64+/-11% and 107+/-2%, respectively). 3. Strong correlations were found between the growth factor-induced reductions in E(max) and their proliferative responses, assessed by [(3)H]-thymidine-incorporation, in BTSM cells. (r=0.97, P=0.002 for methacholine and r=0.93, P=0.007 for KCl). 4. The PDGF-induced reduction in E(max) was inhibited completely by combined treatment with either PD 98059 (30 micro M) or LY 294002 (10 micro M). 5. These results indicate that serum and growth factors may cause a functional shift towards a less contractile phenotype in intact BTSM, which is associated with their proliferative response and dependent on signalling pathways involving the mitogen-activated protein kinase pathway and the phosphatidylinositol-3-kinase pathway.     

Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.

1. In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BB (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stimulated the tyrosine phosphorylation and activation of both pp42 and pp44 kDa forms of mitogen-activated protein (MAP) kinase. 2. Both ET-1 and PDGF stimulated a sustained activation of MAP kinase whilst the response to BK was transient. 3. Activation of MAP kinase occurred in a concentration-dependent manner (EC50 values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml-1). 4. Pretreatment with the protein kinase C (PKC) inhibitor Ro-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activation at 60 min. 5. Following chronic phorbol ester pretreatment, BK-stimulated activation of MAP kinase was abolished whilst the responses to PDGF and ET-1 were only partly reduced (80 and 45% inhibition respectively). 6. Pretreatment with pertussis toxin reduced ET-1 stimulated activation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7. ET-1 also stimulated a 3 fold increase in [3H]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 fold increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8. These results suggest that a pertussis toxin-sensitive activation of MAP kinase may play an important role in ET-1-stimulated DNA synthesis but that activation of MAP kinase alone is not sufficient to induce the magnitude of DNA synthesis observed in response to PDGF.     

Imidazole-induced potentiation of the contractile response to various agonists in vascular smooth muscle

In isolated rabbit aortae, imidazole (10(-4)M) caused a unique nonspecific potentiation of the contractile responses to prostaglandins, norepinephrine, 5-hydroxytryptamine, histamine and potassium only at low concentrations. Imidazole had no effect on the dose-response relationship for Ca2+ in the presence of potassium, 40 mM. Imidazole potentiated contractions in supersensitive strips pretreated with reserpine or with low temperature (5 degrees C) for 5 days. In the presence of theophylline (3 x 10(-4)M), imidazole failed to potentiate the responses to norepinephrine, 5-hydroxy-tryptamine, histamine and potassium, but significantly potentiated responses to prostaglandin E1, E2 and F2 alpha. Imidazole (10(-4)M) significantly decreased the 45Ca uptake of aorta in a Ca-free solution, whereas the drug did not affect 45Ca uptake in a normal solution. These results suggest that imidazole-induced potentiation is related to an increase in Ca2+ permeability and possibly to an additive effect on Ca2+ binding.     

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle

1. The muscarinic receptor subtype involved in the methacholine-induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2-selective antagonist AF-DX 116 and the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide, in addition to the M1-selective antagonist pirenzepine, in a classical Schild analysis. 2. All the antagonists shifted the methacholine dose-response curve to the right in a parallel and concentration-dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF-DX 116 and 4-DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3. The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.     

Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle.     

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.