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.     

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)     

Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(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.     

The inhibitory effects of cromakalim and its active enantiomer BRL 38227 against various agonists in guinea pig and human airways: comparison with pinacidil and verapamil.

The effects of the potassium channel activators, cromakalim, BRL 38227 and pinacidil, and the calcium antagonist, verapamil, have been compared against various spasmogens on airway responses in vitro and in vivo in the guinea pig and also in human isolated bronchi. In guinea pig tracheal spirals, potassium channel activators generally had a greater inhibitory effect than verapamil against tone induced by a wide range of spasmogens (spontaneous, 5-hydroxytryptamine, leukotriene D4, prostaglandin E2). The potassium channel activators had very little effect against potassium chloride- and carbachol-induced tone in guinea pig tracheal spirals [e.g., cromakalim (20 microM) induced relaxations of 0.21 +/- 0.03 (relative to an isoprenaline maximum = 1.0, mean +/- S.E.M.) against carbachol, compared to 0.77 +/- 0.03 against histamine]. In vivo, the potassium channel activators prevented histamine and 5-hydroxytryptamine-induced bronchoconstrictions, but had little inhibitory effect against acetylcholine. In contrast, in human bronchi, cromakalim was capable of inducing powerful concentration-dependent relaxations against carbachol-induced tone [cromakalim (20 microM) induced relaxations of 0.77 +/- 0.09 (relative to isoprenaline = 1.0, mean +/- S.E.M.) against carbachol, compared to 0.95 +/- 0.04 against histamine]. In human bronchi, all the inhibitory agents were more potent and more effective, except that verapamil did not have an increased maximum response. We conclude that potassium channel activators should be effective at relaxing contractions induced by a wide range of spasmogens in man.     

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.     

Low dose verapamil and nitroprusside distinguish tonic from cholinergic-induced contractions in in vivo feline lower esophageal sphincter

Although in vitro studies suggest that cholinergic induced contractions and spontaneous tone are mediated by two different cellular pathways, this has not been shown in vivo. The purpose of this study was to distinguish these types of contractions in the feline lower esophageal sphincter (LES) through use of i.v. selective pharmacologic antagonists. Continuous infusion of the calcium channel antagonist verapamil (16-128 micrograms/kg/min) caused a dose-dependent inhibition of bethanechol induced and tonic pressure change. At 32.0 micrograms/kg/min, verapamil produced significantly greater inhibition of bethanechol induced contractions when compared with inhibition of basal LES tone (57.4 versus 4.8%, P less than .02, n = 5). Verapamil also caused a parallel decrease in LES spike activity associated with bethanechol induced contractions. In contrast, lower doses of nitroprusside (0.5 micrograms/kg/min) inhibited LES tone by over 50% (50.6 +/- 21.8%) yet had no significant effect (16.8 +/- 23.4%) on bethanechol induced contractions. Also, inhibition of basal LES tone correlated poorly with its effect on associated spike activity. It is concluded that in the in vivo feline LES, spike dependent cholinergic contractions can be distinguished from LES tone by the use of low dose verapamil and nitroprusside, respectively. This selective inhibition suggests that two cellular mechanisms mediate LES contractions and that these pathways can be distinguished pharmacologically in vivo.     

Influence of cartilage on reactivity and on the effectiveness of verapamil in guinea pig isolated airway smooth muscle

The authors have examined the effects of cartilage removal on smooth muscle reactivity and the action of verapamil in guinea pig trachealis. In preparations devoid of cartilage, smooth muscle reactivity to both histamine and KCl was reduced. Reactivity to methacholine was unaffected by cartilage removal. In the absence of cartilage, verapamil had a greater depressant effect on the maximum responses to histamine and methacholine than in intact tissues. Similarly, verapamil was more potent against histamine- and methacholine-induced responses in the absence of cartilage where a greater shift to the right was seen in the concentration-response curves when compared with cartilage-containing controls. The spasmolytic action of verapamil on methacholine-induced responses was greater in the absence of cartilage and was greater than its antispasmogenic activity against methacholine (whether or not cartilage was present). Thus, cartilage removal reduces muscle reactivity and increases the potency of verapamil in guinea pig trachealis.     

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)     

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)     

Functional innervation of pig tracheal smooth muscle: neural and non-neural mechanisms of relaxation.

In pig tracheal smooth muscle (TSM), the isometric tension responses to electrical field stimulation (EFS) were compared to exogenous peptides, vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP), as well as to the nicotinic agonist dimethylphenyl piperazinium chloride (DMPP). The objectives of this study were to understand the mechanisms of nonadrenergic, noncholinergic relaxations of pig TSM and identify some putative candidate substances mediating the neural inhibitory response. In strips of TSM obtained from 6- to 12-week-old pigs set up in vitro in organ baths in Kreb's solution, EFS resulted in frequency-dependent contractions that were abolished by 1 microM atropine or 0.1 microM tetrodotoxin (TTX). Addition of DMPP (20-100 microM) resulted in a transient, atropine-sensitive contraction, which quickly desensitized. EFS failed to elicit any further contractions, but the tissues responded to carbachol. In tissues exposed to DMPP and contracted with carbachol, EFS elicited frequency-dependent relaxations that were unaffected by 1 microM propranolol, abolished by TTX and partially inhibited by omega-conotoxin. At the peak of carbachol-induced contraction, addition of DMPP resulted in rapid relaxation reversing spontaneously to base line, with no significant relaxation to further addition of DMPP. DMPP-induced relaxations were unaffected by TTX or omega-conotoxin. These tissues readily relaxed to 10(-8) M VIP. In carbachol precontracted tissues, VIP elicited concentration-dependent relaxations that quickly desensitized. However, these tissues readily relaxed to DMPP. CGRP produced only weak relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine

The influence of the endothelium on smooth muscle tone and the response of the pig right coronary artery to norepinephrine (NE) was studied. Isolated rings of artery with and without endothelium were stretched in the presence of nitroprusside to a tension previously determined to be optimal for contraction. During wash out of the nitroprusside, rings without endothelium spontaneously generated tone representing 24% of the contraction caused by potassium (120 mM); in rings with endothelium no significant spontaneous tone was observed. Relaxations were caused by NE in rings with endothelium contracted with prostaglandin F2 alpha (PGF2 alpha). In rings without endothelium, NE relaxed spontaneously generated tone as well as that produced by PGF2 alpha. Independent of the mode or degree of contraction, rings with endothelium were more sensitive to NE than rings without endothelium. The difference in sensitivity to NE between rings with and without endothelium was likely due to endothelial cell alpha-2 adrenoceptors, inasmuch as the difference was abolished by rauwolscine. In the presence of propranolol and prazosin, endothelium-dependent relaxations were observed which were also inhibited by rauwolscine. Nevertheless, beta adrenoceptors are the predominant mediator of the relaxation to NE of pig coronary smooth muscle, because propranolol caused a greater shift to the right of the relaxation induced by NE compared to that caused by endothelium removal. Accordingly, under resting conditions, NE caused contractions only in the presence of propranolol. These contractions were attenuated by prazosin or rauwolscine, but blocked only by a combination of both alpha adrenoceptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

An in vitro quantitative analysis of the alpha adrenoceptor partial agonist activity of dobutamine and its relevance to inotropic selectivity

In rat anococcygeus muscle, dobutamine produced concentration-related submaximal contractions which were antagonized competitively by phentolamine (pKB = 8.3) and dobutamine antagonized norepinephrine-induced contractions in a competitive manner with an equilibrium dissociation constant for the alpha adrenoceptor of 20 nM (pKB = 7.7). Therefore, dobutamine satisfied criteria for a partial agonist of alpha adrenoceptors having an affinity for alpha adrenoceptors 25 times that of norepinephrine (pKA = 6.3) in this tissue. An estimate of the relative efficacy of dobutamine showed one-fortieth the the efficacy of norepinephrine at the alpha adrenoceptors. Dobutamine contracted rabbit aorta and produced concentration-related relaxations at 1000 times greater concentrations after alkylation of alpha adrenoceptors by phenoxybenzamine. In noncontracted canine saphenous vein, dobutamine had no visible agonist activity but did produce contractions after propranolol. In partially contracted saphenous vein, dobutamine produced a small contraction which was converted to a propranolol-sensitive relaxation of tone after phentolamine. Dobutamine was a full beta adrenoceptor agonist in guinea-pig trachea under spontaneous tone but a partial agonist after strong contraction by bethanechol. This allowed measurement of the pKB of dobutamine at beta adrenoceptors (pKB = 5.35) and estimation of efficacy at beta adrenoceptors relative to isoproterenol (eDob/eIso = 1/20). No evidence for beta adrenoceptor selectivity was found in studies of potency ratios and relative efficacy using isoproterenol for comparison. Dobutamine showed a slight (2-fold) selectivity for inotropy in vitro when compared to isoproterenol in guinea-pig right and left atria. This selectivity was removed by phentolamine suggesting a cardiac alpha-like adrenoceptor effect; this finding was confirmed in propranolol-treated guinea-pig left atria. These results are discussed in terms of the in vivo effects of dobutamine and its use as a tool for classification of beta adrenoceptors, particularly the putative presynaptic beta adrenoceptor.     

Inhibitors of neutral endopeptidase potentiate electrically and capsaicin-induced noncholinergic contraction in guinea pig bronchi

To evaluate the role of airway neutral endopeptidase (NEP) in the regulation of contraction of airway smooth muscle in response to endogenous tachykinins, we studied the effects of the NEP inhibitor phosphoramidon on contractions of guinea pig bronchial smooth muscle strips induced by either electrical field stimulation (EFS) or by capsaicin. In the presence of atropine (10(-6) M), propranolol (10(-6) M), phentolamine (10(-5) M), indomethacin (10(-6) M) and pyrilamine (5 x 10(-6) M) EFS (biphasic; pulse width, 1.0 msec; frequency 0.5-5 Hz for 30 sec; intensity, 20 V) produced noncholinergic, nonadrenergic muscle contraction in a frequency-dependent fashion (P less than .001). Phosphoramidon potentiated the contractile responses to EFS (P less than .01). Leucine-thiorphan (10(-5) M), another NEP inhibitor, potentiated EFS-induced contraction in a similar fashion as phosphoramidon (186 and 182% of control, respectively; each comparison, P less than .025). Captopril, bestatin, leupeptin and physostigmine (each drug, 10(-5) M) were without effect (P greater than .5, N = 5). Capsaicin (1.5 x 10(-8) M) produced long-lasting atropine-resistant smooth muscle contraction, an effect potentiated by phosphoramidon (10(-5) M (P less than .001). Removal of the epithelium slightly but significantly (P less than .05) increased the contractile responses to capsaicin and to EFS at impulse frequencies of 2 and 5 Hz, and phosphoramidon substantially increased contractions in tissues without epithelium. The trachea, bronchi and lungs each contained significant NEP activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of pinacidil and levcromakalim on the contractions elicited electrically or by noradrenaline in the portal vein of the rabbit

The present study was undertaken to examine the antivasoconstrictor effects of pinacidil and levcromakalim, two potassium channel openers (PCOs), on the isolated rabbit portal vein and to define the role for different subtypes of pre- and/or post-synaptic K+ channels in the antivasoconstrictor action of the PCOs. The vein strips were contracted by electrical field stimulation (EFS) or by exogenous noradrenaline (NA). The results of this study showed that pinacidil produced a more potent inhibition of the neurogenic contractions (pD2 = 6.04 +/- 0.05) than of contractions induced by exogenous NA (pD2 = 4.90 +/- 0.10). Glibenclamide (1 microM), a selective blocker of adenosine triphosphate (ATP)-sensitive K+ channels (K(ATP)), did not affect the pinacidil-induced inhibition of contractions evoked by exogenous NA. In contrast, glibenclamide (0.1-10 microM) significantly antagonized the effect of pinacidil on EFS evoked contractions in a noncompetitive manner. There was no difference between the inhibitory effects of levcromakalim on neurogenic contractions (pD2 = 7.58 +/- 0.05) and contractions evoked by exogenous NA (pD2 = 7.64 +/- 0.08). Glibenclamide (1 microM) antagonized in the same manner the levcromakalim-induced inhibition of neurogenic contractions and contractions evoked by exogenous NA. Moreover, glibenclamide competitively antagonized the effect of levcromakalim on EFS induced contractions of the rabbit portal vein (pA2 = 6.40 +/- 0.10). Charybdotoxin (0.4 microM) and apamin (0.1 microM) did not influence the inhibitory effects of pinacidil and levcromakalim, both on contractions evoked by EFS and contractions evoked by exogenous NA. These results suggest that the antivasoconstrictor effect of levcromakalim might be postsynaptic and associated with opening of the smooth muscle K(ATP) channels. In contrast, it is hypothesized that the effect of pinacidil on neurogenic contractions is due to an interference with K(ATP) channels in the neuromuscular synapse. It seems that the action of pinacidil on the NA contractions is mediated by another still undefined mechanisms of pinacidil.     

Effect of different ozone concentrations on the neurogenic contraction and relaxation of guinea pig airways

Summary— Prejunctional and postjunctional effects of several ozone (O₃) concentrations, including those found in highly polluted cities, were evaluated in guinea pig airways. Animals bred in O₃-free conditions were exposed to air or O₃ (0.3, 0.6 or 1.2 ppm) during 4 h, and studied 16–18 h later. Tracheal and bronchial rings were studied in organ baths. Electrical field stimulation (EFS) (100 V, 2 ms, 10 s) was given at increasing frequencies (0.25–16 Hz). Some tissues received atropine (2 μM) and/or propranolol (10 μM). Concentration-response curves to carbachol, isoproterenol, nitroprusside, and substance P were constructed. In tracheas, almost all O₃ concentrations decreased the relaxation at low EFS frequencies, but had no effect on the propranolol-resistant (i-NANC) relaxation, suggesting that only adrenergic relaxation was affected. This was a prejunctional effect, since O₃ did not modify the responses to isoproterenol. Relaxation induced by a nitric oxide (NO) donor, nitroprusside, was not affected by O₃, which agrees with the lack of O₃-effect on i-NANC system. O₃ did not modify the EFS-induced e-NANC contraction in atropine-treated bronchi, nor the contraction caused by exogenous substance P. By contrast, in bronchi without atropine, 1.2 ppm O₃ increased the e-NANC contraction induced by the highest EFS (16 Hz). O₃ increased the maximum responses to carbachol in tracheas (1.2 ppm) and bronchi (0.6 and 1.2 ppm). In conclusion, we found that: a) O₃ decreased adrenergic relaxation in guinea pig tracheas at low EFS frequencies through a prejunctional alteration; b) O₃ did not modify the i-NANC relaxation in tracheas, at least the NO-mediated; c) O₃ added a cholinergic component to the bronchial slow-phase (e-NANC) contraction evoked by EFS; and d) O₃ enhanced the cholinergic responses in trachea and bronchi by a postjunctional mechanism.     

In Vivo Demonstration of Nonadrenergic Inhibitory Innervation of the Guinea Pig Trachea

To determine if electrical stimulation of autonomic nerves could excite nonadrenergic inhibitory motor pathways in the guinea pig respiratory system in vivo, we studied the effects of electrical stimulation of the cervical vagi and sympathetic nerve trunks on pressure changes (P_p) within an isolated, fluid-filled cervical tracheal segment which reflected changes in trachealis muscle tone. We preserved the innervation and circulation of the segment as evidenced by a rise in P_p with vagus nerve stimulation and a fall in P_p with intravenous isoproterenol.     

Vasospasm Contributes to Monosodium Glutamate-lnduced Headache

Consumption of monosodium glutamate has long been considered to precipitate headaches in susceptible patients. In this study the direct effects of glutamate and its metabolite, glutamine, on arterial contractility were examined using rings of rabbit aorta. In a high concentration glutamate caused significant concentration-dependent contractions (EC50, 10(-1)M; maximum tension, 188.4 +/- 33.3 mg wt tension/mg tissue). Agonists and antagonists for alpha-adrenergic, histaminergic, serotonergic, cholinergic, and GABA-nergic receptors as well as inhibition of prostaglandin synthesis failed to influence glutamate contractions. At high concentrations (10(-5)M) the calcium channel blocker, verapamil, inhibited the glutamate response. Glutamate and glutamine both exhibited concentration dependent relaxation of norepinephrine (NE), phenylephrine (PE), histamine, serotonin (5-HT), and prostaglandin F2 alpha (PGF2 alpha)-induced contractions. Kainic acid (10(-4)M), an agonist of one subpopulation of central glutamate receptor, potentiated glutamate-induced vasoconstriction; a higher concentration (10(-3)M) produced an irreversible inhibition of glutamate contractility. Only the central glutamate receptor antagonist, ketamine (10(-4)-10(-2)M), induced a reversible, concentration dependent inhibition of glutamate-induced contractions. Glutamate contractility was not dependent on extracellular calcium, an intact endothelium or neuronal function. These results demonstrate a direct effect of glutamate on peripheral arterial tone. Dietary consumption of large quantities of MSG may represent a serious health hazard to certain individuals with pre-existing vascular disease.     

LY171883, 1-less than 2-hydroxy-3-propyl-4-less than 4-(1H-tetrazol-5-yl) butoxy greater than phenyl greater than ethanone, an orally active leukotriene D4 antagonist

LY171883, 1-less than2-hydroxy-3-propyl-4-less than 4-(h-tetrazol-5-yl)butoxy greater than phenyl greater than ethanone, proved to be a potent antagonist of leukotriene (LT) D4 in guinea-pig ileum, trachea and lung parenchyma. The compound had little or no effect on contractions of isolated tissues to LTB4, prostaglandin F2 alpha, serotonin, histamine, bradykinin or carbamycholine. Responses of trachea to U46619, a thromboxane A2 mimetic, were antagonized by LY171883, but the doses required were approximately 10-fold higher than those necessary to produce the same degree of antagonism against LTD4. U46619 produced weak ileal contractions that were not blocked by LY171883. LY171883 antagonized both LTD4- and antigen-induced increases in total pulmonary resistance in anesthetized guinea pigs. LTD4 given intradermally to guinea pigs caused vascular leakage which was suppressed by prior administration of LY171883. LTC4-induced contractions of isolated ilea were only minimally antagonized by LY171883 whereas this agent reduced LTC4-evoked increases in total pulmonary resistance. Trachea contracted by LTD4 were relaxed by LY171883. Likewise, trachea contracted by either histamine or carbamylcholine were relaxed by LY171883 suggesting that this compound has airway smooth muscle relaxing properties. In vivo experiments supported these observations. In concert with these findings, biochemical studies showed LY171883 to be a potent inhibitor of phosphodiesterase obtained from human polymorphonuclear leukocytes and various guinea-pig tissues. This pharmacologic analysis indicates that LY171883, or a congener, may be of therapeutic value in asthma and in disease states characterized by an overproduction of LTD4.