Inhibitory effect of methacholine on drug-induced relaxation, cyclic AMP accumulation, and cyclic AMP-dependent protein kinase activation in canine tracheal smooth muscle

Functional antagonism between bronchoconstricting and bronchodilating pathways was examined in canine tracheal smooth muscle. Trachealis strips were contracted with either 0.3 microM (EC55) or 3.0 microM (EC80) methacholine before being relaxed by the cumulative addition of isoproterenol, prostaglandin E2, or forskolin. The EC50 for all three relaxants was increased 10-fold in tissues contracted with 3.0 microM methacholine vs. those contracted with 0.3 microM methacholine. Moreover, contracting tissues with the higher concentration of methacholine reduced the maximum relaxation induced by prostaglandin E2 and isoproterenol. Forskolin produced total relaxation regardless of the concentration of methacholine used and thus was a much more effective bronchodilator than either isoproterenol or prostaglandin E2. The inhibitory effect of methacholine on the relaxant response to these agents was paralleled by a reduction in drug-stimulated cyclic AMP-dependent protein kinase activity. Methacholine reduced the maximum activation of cyclic AMP-dependent protein kinase elicited by isoproterenol, prostaglandin E2 and submaximal concentrations of forskolin, which was a much more powerful enzyme activator than the other two agents. The ability of a maximum concentration of forskolin (30 microM) to activate cyclic AMP-dependent protein kinase was not inhibited by methacholine. Although methacholine also appeared to suppress drug-stimulated cyclic AMP accumulation, the inhibitory effect was only statistically significant in forskolin-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional differences in relaxation of electric field-stimulated canine airway smooth muscle by verapamil, isoproterenol and prostaglandin E2

Regional differences in contraction produced by methacholine and electric field stimulation (EFS) and in relaxation produced by isoproterenol, prostaglandin E2 and verapamil were studied in isolated canine airway smooth muscle in vitro. Low-frequency EFS (3 Hz, 0.5 msec, 50 V) contracted thoracic trachealis to 43% of maximal EFS response, whereas cervical trachealis contracted to only 14% of maximum. EFS at 10 Hz produced 75% of the maximal response in both regions of the trachea. These EFS responses were abolished by 0.1 microM tetrodotoxin and 1.0 microM atropine. Contraction produced by EFS was also matched in each tissue by contraction with methacholine. The concentrations of methacholine that matched EFS at 10 Hz were 52 +/- 7, 378 +/- 84 and 66 +/- 11 nM for cervical and thoracic trachealis and lobar bronchi, respectively. Both EFS and matched methacholine contractions of cervical trachealis and lobar bronchi were completely relaxed by isoproterenol, whereas thoracic trachealis relaxed maximally to only 60% of induced tone. When verapamil was used to relax EFS and matched methacholine contractions, cervical trachealis was completely relaxed whereas thoracic trachealis relaxed to 15% of induced tone. Although there was a regional difference in the relaxant potency of isoproterenol and, to some extent, verapamil, there was no difference in isoproterenol or verapamil EC50 values for EFS vs. matched methacholine contractions within each region. In contrast, EFS contractions of thoracic trachealis were more sensitive to prostaglandin E2-induced relaxation than were matched methacholine contractions. These data demonstrate marked differences in cholinergic and beta adrenergic receptor-mediated responses between regions of the tracheobronchial tree.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-related differences in the relationship between cAMP content and protein kinase activity in canine trachealis.

We investigated the relationships between relaxation, cyclic AMP (cAMP) accumulation and cAMP-dependent protein kinase (cAMP-PK) activity in canine tracheal smooth muscle. In time course and concentration-response studies, forskolin and isoproterenol elicited relaxation of isolated trachealis strips that was accompanied by an increase in cAMP content and an activation of cAMP-PK. Although these results were consistent with the proposal that cAMP is a second messenger mediating relaxation of airway smooth muscle, close inspection of the data revealed a discrepancy in the relationship between cAMP accumulation and relaxation. To induce equivalent degrees of tracheal relaxation, forskolin generated greater increments in cAMP accumulation than did isoproterenol. On the other hand, the activation state of cAMP-PK correlated reasonably well with relaxation regardless of which agonist was used. Further analysis of the data revealed that the apparent disparity between cAMP accumulation and relaxation could largely be explained at the level of the relationship between cAMP content and cAMP-PK activity: compared to isoproterenol, forskolin induced greater increases in cAMP accumulation to achieve the same activation state of cAMP-PK. These observations lend support to the proposal that in canine trachealis, various components of the cAMP/cAMP-PK cascade exist in distinct subcellular compartments such that not all of the cAMP generated in response to forskolin has access to its target enzyme, cAMP-PK.     

Functional antagonism in canine tracheal smooth muscle: inhibition by methacholine of the mechanical and biochemical responses to isoproterenol

The biochemical basis for the functional interaction between bronchoconstricting and bronchodilating pathways was investigated. Contracting canine trachealis strips with increasing concentrations of methacholine resulted in a progressive shift to the right of isoproterenol concentration-response curves. Thus, the EC50 for the relaxant response to isoproterenol was nearly 500-fold higher in preparations exposed to 3.0 microM methacholine than in tissues exposed to 0.03 microM methacholine. The maximum relaxation produced by isoproterenol was also dependent upon the initial muscarinic cholinergic tone. For example, isoproterenol reversed completely the contraction induced by 0.03 microM methacholine but did not relax trachealis strips contracted with 30 microM methacholine. To identify the molecular mechanism responsible for this functional antagonism, experiments were conducted to determine the effect of methacholine on isoproterenol-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation. Methacholine did not alter basal cyclic AMP content but did reduce cyclic AMP accumulation in response to isoproterenol. Furthermore, the ability of isoproterenol to activate cyclic AMP-dependent protein kinase was inhibited by methacholine in a concentration-dependent manner. This inhibition paralleled the decrease in mechanical responsiveness to isoproterenol. These results suggest that muscarinic cholinergic stimulation of canine tracheal smooth muscle functionally antagonizes the relaxant responses to beta adrenergic agonists and that a portion of this antagonism may be due to a suppression of catecholamine-stimulated cyclic AMP accumulation and cyclic AMP-dependent protein kinase activation.     

Relationship between cyclic guanosine monophosphate accumulation and relaxation of canine trachealis induced by nitrovasodilators.

The role of cyclic GMP (cGMP) in mediating relaxation of canine trachealis produced by nitrovasodilators (NVDs), compounds that activate guanylate cyclase, was examined. Sodium nitroprusside (SNP) produced a concentration-dependent relaxation of the canine trachealis that was accompanied by a concentration-related increase in cGMP content. In time course studies, relaxation of isolated trachealis strips induced by 30 microM SNP was paralleled by an increase in cGMP that reached a maximum of 18-fold above basal levels within 2 min. Zaprinast, an inhibitor of the cGMP-specific phosphodiesterase, potentiated both SNP-induced relaxation and cGMP accumulation. A cell-permeable analog of cGMP, 8-bromo-cGMP, mimicked the relaxant effects of SNP. Also assessed were the effects of methylene blue, an agent that inhibits soluble guanylate cyclase activity, and hemoglobin, an agent that competitively binds NO-containing compounds. In these experiments, tissues were pretreated with the above agents for 10 min, contracted with 1 or 3 microM methacholine, and then relaxed by the cumulative addition of SNP or two other NVDs, S-nitroso-N-acetyl-penicillamine (SNAP) and glyceryl trinitrate (GTN). Tissues were flash-frozen after adding the final concentration of the various NVDs and assayed for cGMP. Methylene blue and hemoglobin suppressed both cGMP accumulation and relaxation in response to SNAP and GTN. in contrast, methylene blue and hemoglobin inhibited SNP-induced cGMP accumulation but, paradoxically, potentiated SNP-induced relaxation. The results of this study generally support a role for cGMP in NVD-induced relaxation of airway smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of cyclic AMP-dependent protein kinase during canine lower esophageal sphincter relaxation

In isolated strips of opossum lower esophageal sphincter (LES) smooth muscle, elevation of cyclic AMP (cAMP) content is associated with relaxation. Because the activation state of cyclic nucleotide-dependent protein kinases may be a more sensitive measure of functionally important changes in cyclic nucleotide levels, we examined the ability of several pharmacological agents and electrical stimulation of the enteric neurons to activate cAMP dependent-protein kinase (cA-PK) and to relax isolated strips of LES smooth muscle. Addition of either isoproterenol or SK&F 94120, a selective inhibitor of the low Km cAMP phosphodiesterase to isolated strips of canine LES produced concentration-dependent increases in the activity ratio of cA-PK and concentration-dependent relaxations of canine LES. In contrast, although both zaprinast (M&B 22948), a selective inhibitor of the cyclic GMP selective phosphodiesterase, and sodium nitroprusside relaxed canine LES neither drug significantly increased the activity ratio of cA-PK. Furthermore, electrical stimulation of the enteric neurons produced a frequency-dependent relaxation but did not significantly activate cA-PK. To eliminate the possibility that the rapid metabolism of cAMP prevented us from observing a significant activation of cA-PK during electrical field stimulation, the ability of electrical field stimulation (1.0 Hz) to activate cA-PK was examined in the presence of 0.1 mM 3-isobutyl-1-methylxanthine. At at concentration of 0.1 mM, 3-isobutyl-1-methylxanthine, by itself, significantly increased the activity ratio of cA-PK; however, there was no additional activation produced by electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular reactivity of isolated thoracic aorta of the C57BL/6J mouse

We characterized the thoracic aorta from the C57BL/6J mouse, a strain used commonly in the generation of genetically altered mice, in response to vasoactive substances. Strips of aorta were mounted in tissue baths for measurement of isometric contractile force. Cumulative concentration-response curves to agonists were generated to observe contraction, or relaxation in tissues contracted with phenylephrine or prostaglandin F(2alpha) (PGF(2alpha)). In endothelium-denuded strips, the order of agonist contractile potency (-log EC(50) [M]) was norepinephrine > phenylephrine = 5-hydroxytryptamine > dopamine > PGF(2alpha) > isoproterenol > KCl. Angiotensin II and endothelin-1 were weakly efficacious (15% of maximum phenylephrine contraction), as were UK14,304, clonidine, histamine, and adenosine. In endothelium-intact strips, agonists still caused contraction and both angiotensin II and endothelin-1 remained ineffective. In experiments focusing on angiotensin II, angiotensin II-induced contraction was abolished by the AT(1) receptor antagonist losartan (1 microM) but was not enhanced in the presence of the AT(2) receptor antagonist PD123319 (0.1 microM), tyrosine phosphatase inhibitor orthovanadate (1 microM) or when angiotensin II was given noncumulatively. Prazosin abolished isoproterenol-induced contraction and did not unmask isoproterenol-induced relaxation. Angiotensin II and endothelin-1 did not cause endothelium-dependent or -independent relaxation in phenylephrine- or PGF(2alpha)-contracted tissues. Acetylcholine but not histamine, dopamine, or adenosine caused an endothelium-dependent vascular relaxation. These experiments provide information as to the vascular reactivity of the normal mouse thoracic aorta and demonstrate that the mouse aorta differs substantially from rat aorta in response to isoproterenol, angiotensin II, endothelin-1, histamine, and adenosine.     

Differential inhibitory effects of forskolin, isoproterenol, and dibutyryl cyclic adenosine monophosphate on phosphoinositide hydrolysis in canine tracheal smooth muscle.

A characteristic feature of airway smooth muscle is its relative sensitivity to relaxant effects of beta adrenergic agonists when contracted by inflammatory mediators, such as histamine, vs. resistance to these relaxant effects when contracted by muscarinic agonists. Because contractions presumably depend upon the hydrolysis of membrane phosphoinositides (PI) and the generation of inositol phosphates (IP), our goal was to test for the effects of forskolin, isoproterenol, and dibutyryl cAMP on histamine- vs. methacholine-induced IP accumulation in canine tracheal smooth muscle. Methacholine (10(-3) M) was a more effective stimulant of IP accumulation (9.6 +/- 2.1-fold increase) than equimolar histamine (3.6 +/- 0.5-fold increase) in this tissue. When responses to equieffective methacholine (4 x 10(-6) M) and histamine (10(-3) M) were compared, neither forskolin, isoproterenol, nor dibutyryl cAMP significantly decreased IP accumulation in response to methacholine. In contrast, each of these three agents significantly decreased responses to histamine (by 56 +/- 9, 52 +/- 2, and 61 +/- 2%, respectively). We concluded that, in canine tracheal smooth muscle, increased cAMP is associated with inhibition of PI hydrolysis in response to histamine but not methacholine. The findings suggest a novel mechanism for selective modulation by cAMP of receptor-mediated cellular activation.     

Differential effects of methacholine and leukotriene D4 on cyclic nucleotide content and isoproterenol-induced relaxation in the opossum trachea

The effects of leukotriene D4 and methacholine on cyclic nucleotide content and isoproterenol-induced relaxation were examined in the isolated opossum trachea. Although leukotriene D4 (-log EC50 = 6.70) was a more potent contractile agent than methacholine (-log EC50 = 5.78), the maximal response to leukotriene D4 was only 65% of the maximum response to methacholine. Contraction of tracheal strips with leukotriene D4 was accompanied by a 3-fold increase in cyclic GMP accumulation. Methacholine-induced contraction was not associated with an increase in cyclic GMP. Neither agent altered basal cyclic AMP content. Additional experiments were carried out to examine functional inhibitory interactions between bronchoconstricting and bronchodilating pathways. In these studies, cumulative isoproterenol concentration-response curves were constructed in tracheal strips contracted with three different concentrations of methacholine and in tissues contracted with three corresponding equieffective concentrations of leukotriene D4. Although the relaxant response to isoproterenol decreased as tissues were contracted with higher concentrations of either agent, the inhibitory effect of methacholine on isoproterenol-induced relaxation was much greater than the inhibitory effect of leukotriene D4. Previous studies from our laboratory suggested that a potential explanation for the greater inhibitory effect of methacholine on the mechanical response to isoproterenol was that methacholine may inhibit isoproterenol-stimulated cyclic AMP accumulation whereas leukotriene D4 may not. However, neither methacholine nor leukotriene D4 inhibited isoproterenol-stimulated cyclic AMP accumulation in the opossum trachea. The results of this study indicate that the sensitivity of airway smooth muscle to beta adrenoceptor agonists is influenced both by the initial contractile state of the tissue and by the type of agent used to induce tone.     

Effects of milrinone on contractile responses of guinea pig trachea, lung parenchyma and pulmonary artery

The effects of milrinone, a bipyridine with known vasodilator activity, on guinea pig tracheal-spirals, lung parenchymal strips and pulmonary artery rings in vitro were compared with the effects of isoproterenol and aminophylline on these tissues. The concentration of milrinone that produced 50% relaxation (IC50) of tracheal spirals constricted by carbachol was 3.6 X 10(-5) M. Isoproterenol (IC50, 9.5 X 10(-8) M) was significantly (P less than .001) more potent and aminophylline (IC50, 1.2 X 10(-4) M) was significantly (P less than .001) less potent than milrinone in this effect. The IC50 for milrinone for lung parenchymal strips contracted by histamine was 3.2 X 10(-5) M, whereas the IC50 for isoproterenol was significantly (P less than .001) less, 1.4 X 10(-7) M; aminophylline produced only limited relaxation of lung parenchymal strips. Milrinone relaxed pulmonary artery rings constricted by norepinephrine with an IC50 of 3.8 X 10(-6) M, whereas neither isoproterenol nor aminophylline produced a 50% relaxation. Pretreatment of tracheal spirals, lung parenchymal strips and pulmonary artery rings with 1.6 X 10(-4) M milrinone inhibited subsequent contraction by carbachol, histamine and norepinephrine, respectively. The relaxant effects of milrinone were not influenced by treatment with atropine, cimetidine, mepyramine, phentolamine or propranolol. However, indomethacin blocked milrinone's relaxant effects on tracheal spirals effectively, but not on pulmonary artery rings or lung parenchymal strips, suggesting distinct modes of action on various tissue types.     

SK&F 104353: selective antagonism of peptidoleukotrine-induced changes in electrolyte transport by rat ileal mucosa in vitro

Effects of the peptidoleukotriene receptor antagonist, SK&F 104353, were examined in vitro using stripped rat ileal mucosa in Ussing chambers. Changes in short-circuit current (Isc), transepithelial conductance (G1) and unidirectional and net Na+ and Cl fluxes measured in the absence or presence of SK&F 104353 (1 microM) revealed that serosal addition produced a decrease in net Na+ absorption and serosal-to-mucosal Cl- flux. Serosal addition of leukotriene (LT)D4 (10 muM) or LTE4 (10 microM) elicited transient increases in Isc and sustained decreases in G1 which were antagonized by serosal addition of SK&F 104353 in a concentration-dependent manner. Mucosal addition of SK&F 104353 (1 microM) also reduced the increase in Isc elicited by LTD4 (10 microM). LTD4 (10 microM) decreased unidirectional and net Na+ and Cl- fluxes along with G1 in the steady state. All of these changes were abolished by pretreatment with SK&F 104353 (1 microM). The intestinal secretagogues prostaglandin F2 alpha, histamine, serotonin, substance P and the thromboxane mimic, U46619, produced changes in Isc qualitatively similar to those of LTD4. However, the transient increase in Isc produced by these secretagogues was not antagonized by SK&F 104353 (1 microM). Additionally, lysbradykinin- and prostaglandin E1-induced increases in Isc were not antagonized by SK&F 104353 (1 microM). Stimulation with histamine (10 microM) or pretreatment with LTB4 (5 microM) did not alter the increase in Isc or decrease in Gt produced by the subsequent addition of LTD4 (10 microM). Pretreatment of the tissues with mepyramine (10 microM) also did not reduce the increase in Isc elicited by LTD4 (10 microM), although it inhibited completely the increase in Isc produced by histamine (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of nonadrenergic neural relaxation in guinea pig airways by a cyclic cAMP phosphodiesterase inhibitor

We have studied the effect of phosphodiesterase inhibitors on relaxation of guinea pig tracheal smooth muscle in an attempt to elucidate the role of cyclic nucleotides in relaxation to stimulation of inhibitory nonadrenergic noncholinergic (i-NANC) nerves. SK&F 94120 (1-10 microM) potentiated relaxation induced by isoproterenol, vasoactive intestinal peptide (VIP) and electrical field stimulation (EFS) in the presence of atropine and propranolol but had no effect on relaxation induced by sodium nitroprusside. Zaprinast (3-30 microM) potentiated relaxation induced by sodium nitroprusside but not by isoproterenol or VIP. A small potentiation of relaxation to EFS was induced by 30 microM zaprinast but not by lower concentrations. Tetrodotoxin attenuated relaxations induced by EFS suggesting that they are at least partly neurogenic in origin. SK&F 94120 and zaprinast had no effect of tetrodotoxin-resistant relaxation to EFS. The guanylate cyclase inhibitor had no effect on EFS-induced relaxation. These findings suggest that cyclic AMP may mediate relaxation of guinea pig tracheal smooth muscle in response to stimulation of i-NANC nerves, and are in agreement with the view that VIP may be the neurotransmitter released by i-NANC nerves in this tissue.     

Relaxant effect of amiloride on canine tracheal smooth muscle

Amiloride, a K+-sparing diuretic, relaxed canine tracheal smooth muscle strips contracted isometrically with high potassium (KCl), carbachol, serotonin and histamine. This indicated that relaxation was not linked to an interaction with an agonist specific receptor. Amiloride-induced relaxation was also not mediated through the production of relaxant prostaglandins, or by the endogenous release of catecholamines. During potassium contractions, amiloride addition produced a slow monophasic, dose-dependent relaxation (IC50 = 12.3 microM). In carbachol contracted strips, 1 and 10 microM amiloride induced a slow monophasic relaxation. With 35 to 250 microM, an initial rapid phase (IC50 = 75.5 microM) was superimposed onto this slow phase (IC50 = 23.5 microM), producing a biphasic relaxation. The rates of relaxation decreased with increased external [Na+] regardless of stimulus, suggesting possible competitive inhibition of a sodium-dependent process. Exposure caused a rapid decline in tension followed by a recovery phase. Tension maintenance during potassium contraction decreased transiently upon the addition of acid to a much lesser extent. Amiloride (100 microM) depressed tension recovery after acid exposure in both cases. Based on the known actions of this drug, inhibition of the Na+-H+ antiporter appears to be consistent with these data. This suggests amiloride may well belong to a new class of smooth muscle relaxants.     

Histamine receptors on human isolated pulmonary arterial muscle preparations: effects of endothelial cell removal and nitric oxide inhibitors.

Human isolated intact pulmonary arterial muscle ring preparations which were precontracted with serotonin (10 microM) relaxed when stimulated with low concentrations of histamine, 2-[2-thiazolyl]ethylamine or 2-[pyridyl]ethylamine (pD2 values: 8.66 +/- 0.22, 7.10 +/- 0.06 and 6.20 +/- 0.26, respectively) or contracted at higher concentrations of these agonists. This relaxant response was obliterated in endothelial denuded tissues. Chlorpheniramine (H1-antagonist; 0.25 and 2.5 microM) induced a small contractile response in the tissues at resting tone (0.08 +/- 0.03 g and 0.10 +/- 0.10 g, respectively). Chlorpheniramine also shifted the histamine relaxation curves to the right (pD2 values: control, 8.85 +/- 0.31; 0.25 microM, 6.90 +/- 0.41; and 2.5 microM, 5.58 +/- 0.30; N = 6). Dimaprit (H2-agonist) induced a small relaxation (20%) in both intact and denuded tissues. Treatment of the tissues with cimetidine (H2-antagonist; 50 microM), burimamide (H2/H3-antagonist; 10 microM) and impromidine (H2-agonist/H3-antagonist; 1 microM) did not alter histamine-induced relaxation or contraction. Indomethacin (1.7 microM) caused a small contraction in these tissues and significantly reduced the histamine relaxation. The nitric oxide inhibitors (L-NG-monoethyl-L-arginine, 30 and 300 microM; or L-NG-nitroarginine, 30 and 300 microM) induced a slight and variable contraction in the preparations. However, these inhibitors, only in the presence of indomethacin, inhibited the relaxant effects of histamine and potentiated the contractions induced by this amine. These data suggest that a dual endogenous vasodilatory mechanism is present in human isolated pulmonary arterial muscle preparations and that products of the cyclooxygenase and endothelium-derived relaxing factor-nitric oxide pathway may interact to regulate histamine stimulation of H1-receptors.     

Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guinea pig and human airways

In this report, we describe the in vitro and in vivo pharmacologic profile of 2(S)-hydroxy-3(R)-[(2-carboxyethyl)thio]-3-[2-(8-phenyloctyl)pheny l]- propanoic acid (SK&F 104353) in guinea pig and human airways. In the isolated guinea pig trachea, SK&F 104353 was a potent, competitive antagonist of leukotriene (LT) D4-induced contractions (pA2 = 8.6). In contrast, SK&F 104353 produced little effect on LTC4 concentration-response curves under conditions where the bioconversion of LTC4 to LTD4 was inhibited. LTE4-induced contractions in guinea pig trachea were sensitive to inhibition by SK&F 104353 (pKB greater than 8.9). SK&F 104353 (10 microM) had no intrinsic contractile activity and was without effect on contractions produced by KCl, histamine, prostaglandin D2, platelet-activating factor or U-44069 in guinea pig trachea. Furthermore, unlike other purported LT antagonists, LT 171883 and FPL 55712, SK&F 104353 (30 microM) did not inhibit cyclic nucleotide phosphodiesterase activity measured in homogenates from canine tracheal smooth muscle. In the isolated human bronchus, SK&F 104353 produced concentration-dependent rightward shifts in LTD4 concentration-response curves and, unlike in guinea pig trachea, was an effective antagonist of LTC4-induced contractions with a pKB of 8.0 to 8.4. This provides further evidence that, in contrast to guinea pig airways, responses produced by LTC4 and LTD4 in human bronchus appear to be mediated via the same LT receptor population. SK&F 104353 was also an effective antagonist of LTE4-induced responses in human bronchus (pKB greater than 8.2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of drug responses in vivo and in vitro in airways of dogs with and without airway hyperresponsiveness

Basenji-greyhound (BG) dogs demonstrate marked nonspecific airway hyperresponsiveness. To assess the possible contribution of an abnormal sensitivity of airway smooth muscle to this phenomenon, we studied the in vitro contractile responses to methacholine and histamine and the relaxant response to isoproterenol in trachealis muscle from five BG dogs with airway hyperresponsiveness in vivo and from five greyhound dogs that served as a control population. Isoproterenol responses were determined against a half-maximal methacholine contraction. Aerosol methacholine concentrations required to produce a 2-fold increase in pulmonary resistance were 0.07 +/- 0.02 (+/- S.E.) mg/ml in BG dogs and 0.67 +/- 0.26 mg/ml in greyhounds; pD2 values for methacholine-induced contraction of cervical trachealis muscle were 7.03 +/- 0.11 in BG dogs and 7.50 +/- 0.11 in greyhounds. A significant (P less than .01) negative correlation was found between methacholine sensitivity in vivo and in vitro. Aerosol concentrations of histamine required to produce a 2-fold increase in pulmonary resistance were 0.19 +/- 0.06 mg/ml in BG dogs and 1.44 +/- 0.43 mg/ml in greyhounds; pD2 values for histamine were identical in BG dogs (4.95 +/- 0.08) and greyhounds (5.05 +/- 0.19). Isoproterenol pD2 values were less in the trachealis muscle (cervical) of BG dogs (6.76 +/- 0.10) than in that of greyhounds (7.93 +/- 0.16), but this is probably a consequence of the higher concentration of methacholine needed to contract BG muscles. We conclude that the airway hyperresponsiveness of BG dogs does not reflect an increased sensitivity of airway smooth muscle per se.     

Platelet-activating factor-acether-induced relaxation of guinea pig airway muscle: role of prostaglandin E2 and the epithelium

A fixed concentration of paf-acether (platelet-activating factor; 4 microM) relaxed isolated guinea pig tracheal preparations which had been contracted with histamine (50 microM), serotonin (1 microM) or leukotriene D4 (0.1 microM). The relaxations were approximately 43, 100 and 57%, respectively. We did not observe any relaxant effect of paf-acether (4 microM) in tissues contracted with acetylcholine (50 microM). Both lyso paf-acether (10 microM) and bovine serum albumin (25 micrograms/ml) were without effect on histamine-contracted preparations. In the presence of indomethacin (1.7 microM; 30 min) or aspirin (0.1 mM; 30 min) the relaxant effect of paf-acether (4 microM) in tissues contracted with histamine was significantly reduced to approximately 10 and 12%, respectively. When paf-acether (4 microM) was added to histamine-contracted tracheal preparations in the presence of noradrenaline (0.1 microM) or prostaglandin E2 (PGE2, 10 nM) the relaxations were 62 and 82%, respectively. Noradrenaline and PGE2 alone had only a slight relaxant effect in these tissues (7 and 14%, respectively). In the presence of indomethacin (1.7 microM) the synergistic effect of paf-acether and PGE2 was still observed. The basal production of PGE2 in isolated guinea pig tracheal preparations was 4.6 +/- 1.4 pg/mg of tissue. In the presence of paf-acether (4 microM) increased levels of this prostanoid were detected (11.2 +/- 2.4 pg/mg of tissue). Isolated guinea pig tracheal preparations when contracted with histamine released PGE2 (17.6 +/- 4.1 pg/mg of tissue). In the presence of histamine and paf-acether there was a significant increase in detectable levels of PGE2 (48.6 +/- 13.2 pg/mg of tissue).(ABSTRACT TRUNCATED AT 250 WORDS)     

Response and sensitivity of isolated human pulmonary muscle preparations to pharmacological agents

The responsiveness (grams per millimeter squared) and sensitivity (pD2 value) of various agonists were examined in isolated stored human bronchial and pulmonary arterial and venous preparations. In isolated bronchial muscles, large preparations (internal diameter about 6 mm) were less responsive (grams per millimeter squared) to contractile agents than smaller preparations (internal diameter approximately 2 mm). Noncumulative concentration-effect curves were produced in bronchial preparations using histamine, acetylcholine, carbachol and barium chloride. Histamine contracted both bronchial and vascular preparations whereas 5-hydroxytryptamine contracted only vascular tissues. The latter effect was always blocked by either methysergide or ketanserin. 5-hydroxytryptamine relaxed bronchial tissues that were contracted with either histamine, acetylcholine or prostaglandin E2. This relaxation was not antagonized by methysergide, ketanserin, propranolol or indomethacin. Dimaprit and 4-methyl histamine were without effect in isolated contracted bronchial preparations. Vasoactive intestinal peptide, Substance P and platelet-activating factor when added to preparations at resting tone failed to induce a contraction. These agents did not relax histamine-contracted isolated human pulmonary muscle preparations. Anti-immunoglobulin E antibody sometimes contracted isolated human bronchial muscle but not pulmonary vascular preparations. However, these data were difficult to assess because of the variations observed. Anti-immunoglobulin G antibody was inactive. Noradrenaline did not elicit a physiological response in isolated bronchial muscle preparations at concentrations which always induced a contraction in the pulmonary vascular preparations. In the presence of propranolol, noradrenaline neither contracted nor relaxed isolated human bronchial preparations. We also determined the sensitivity of isolated bronchial muscle preparations to isoproterenol, salbutamol and theophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses mediated via beta-1 adrenoceptors but not beta-2 adrenoceptors exhibit supersensitivity after chronic reserpine pretreatment

The purpose of this study was to examine whether reserpine pretreatment induces supersensitivity of both beta-1 and beta-2 adrenoceptor-mediated responses. Guinea pigs received reserpine (0.5 mg kg-1 s.c. or i.p.) daily for 7 days. Isolated tissues were set up in the presence of phentolamine (5 microM) and metanephrine (10 microM) and the sensitivity to isoproterenol and, where possible, a partial agonist (ritodrine, salbutamol or prenalterol) was determined. The beta adrenoceptor-mediated responses were recorded as the increase in rate and tension of right and left atria, inhibition of carbachol-induced contractions of ileum, relaxation of aortic spirals contracted with histamine, inhibition of transmurally stimulated vas deferens and relaxation of tracheal spirals and lung strips with intrinsic tone. The atria exhibited supersensitivity after reserpine pretreatment (s.c. and i.p.) as a leftwards shift of the isoproterenol concentration-response curve and elevation of the prenalterol maximum response. The ileum was also supersensitive, but only when tissues from animals receiving i.p. reserpine were compared with shams, which themselves were subsensitive or when reserpine was administered s.c. The trachea was also supersensitive, but not the aorta, lung and vas deferens, the responses of which are mediated via beta-2 adrenoceptors. In contrast, beta-1 adrenoceptors are involved in the atrial, ileal and tracheal responses. Therefore, only responses mediated via beta-1 adrenoceptors exhibited reserpine-induced supersensitivity which supports the hypothesis that beta-1 but not beta-2 adrenoceptors receive a sympathetic innervation.     

The bronchopulmonary pharmacology of SK&F 104353 in anesthetized guinea pigs: demonstration of potent and selective antagonism of responses to peptidoleukotrienes

The bronchopulmonary pharmacology of SK&F 104353 [2(S)-hydroxy-3(R)-[2(2-carboxyethyl)thio]-3[2-(8- phenyloctyl)phenyl]-propanoic acid], a potent and selective leukotriene (LT) receptor antagonist in vitro, was assessed in anesthetized, spontaneously breathing guinea pigs. Aerosol administration of SK&F 104353 (5-2000 micrograms/ml x 100 breaths) reduced in a concentration-dependent manner the response to a standard LTD4 challenge (4.33 micrograms/ml x 5 breaths) given 30 min later. Inhalation of a 2000 micrograms/ml solution abolished LTD4-induced bronchoconstriction for at least 2 hr. The i.v. administration of SK&F 104353 10 min before challenge antagonized LTD4-induced bronchoconstriction with an ID50 of 0.55 mumol/kg (0.25 mg/kg). Substantial antagonism of LTD4-induced bronchospasm was observed for at least 60 min after i.v. administration of 5 mumol/kg of SK&F 104353. Infusion of SK&F 104353 at various rates revealed that a steady-state plasma concentration of 0.125 microM (0.06 micrograms/ml) reduced LTD4-induced bronchoconstriction by 60%. In addition to preventing the response to LTD4, i.v. administered SK&F 104353 (10 mumol/kg) rapidly and completely reversed ongoing LTD4-induced bronchoconstriction. SK&F 104353 also was effective when given intraduodenally 1 hr before LTD4 challenge, although the ID50 (32 mumol/kg) was 60-fold greater than the i.v. ID50. Given intragastrically, 100 mumol/kg of SK&F 104353 abolished the response to LTD4 for 1 hr, and reduced the response for 6 hr. SK&F 104353 (20 mumol/kg i.v.) had no effect on the bronchoconstriction induced by aerosolized acetylcholine, histamine or U-44069, but did antagonize the response to LTC4. SK&F 104353 alone did not produce bronchoconstriction when administered by any route or dose.(ABSTRACT TRUNCATED AT 250 WORDS)