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.     

Hyperosmolar solution effects in guinea pig airways. IV. Lipopolysaccharide-induced alterations in airway reactivity and epithelial bioelectric responses to methacholine and hyperosmolarity

We investigated the in vivo and in vitro effects of lipopolysaccharide (LPS) treatment (4 mg/kg i.p.) on guinea pig airway smooth muscle reactivity and epithelial bioelectric responses to methacholine (MCh) and hyperosmolarity. Hyperosmolar challenge of the epithelium releases epithelium-derived relaxing factor (EpDRF). Using a two-chamber, whole body plethysmograph 18 h post-treatment, animals treated with LPS were hyporeactive to inhaled MCh aerosol. This could involve an increase in the release and/or actions of EpDRF, because LPS treatment enhanced EpDRF-induced smooth muscle relaxation in vitro in the isolated perfused trachea apparatus. In isolated perfused tracheas the basal transepithelial potential difference (Vt) was increased after LPS treatment. The increase in Vt was inhibited by amiloride and indomethacin. Concentration-response curves for changes in Vt in response to serosally and mucosally applied MCh were biphasic (hyperpolarization, <3 x 10(-7)M; depolarization, >3 x 10(-7)M); MCh was more potent when applied serosally. The hyperpolarization response to MCh, but not the depolarization response, was potentiated after LPS treatment. In both treatment groups, mucosally applied hyperosmolar solution (using added NaCl) depolarized the epithelium; this response was greater in tracheas from LPS-treated animals. The results of this study indicate that airway hyporeactivity in vivo after LPS treatment is accompanied by an increase in the release and/or actions of EpDRF in vitro. These changes may involve LPS-induced bioelectric alterations in the epithelium.     

Muscarinic stimulation of adenylate cyclase activity of rat olfactory bulb. I. Analysis of agonist sensitivity

In rat olfactory bulb, stimulation of muscarinic receptors activates adenylate cyclase. In the present study we have examined a variety of muscarinic receptor stimulants to characterize the agonist profile of this response. Analysis of agonist concentration-response curves revealed the following rank order of potency: oxotremorine-M greater than oxotremorine greater than BM5 greater than acetylcholine greater than carbachol = methacholine greater than (+/-)muscarine greater than arecoline greater than pilocarpine greater than RS 86 greater than McN-A-343 greater than bethanechol. Acetylcholine, oxotremorine-M, carbachol, (+/-)muscarine and metacholine behaved as full agonists, whereas the other stimulants displayed lower efficacies. The slope values of the concentration-response curves were close or equal to 1, except those of the carbachol and pilocarpine curves, which showed values significantly lower than 1. Moreover, the slope of the pilocarpine curve was differentially changed by the M1 antagonist pirenzepine and the M2 antagonist AF-DX 116. The agonist profile of the muscarinic stimulation of adenylate cyclase in the olfactory bulb correlated well with that exhibited by the muscarinic inhibition of the enzyme activity in the striatum, suggesting that the two responses are mediated by a similar receptor subtype. Sodium ion modulated the agonist affinity for both adenylate cyclase-coupled receptor systems.     

Cardiac responsiveness to alpha and beta adrenergic amines: effects of carbachol and hypothyroidism

Previous reports of cardiac beta to alpha adrenoceptor interconversion secondary to hypothyroidism left open the alternative possibility of a functional influence by hypothyroidism on the inotropic and chronotropic effects of adrenergic amines through a different mechanism. To test this possibility, the effects of hypothyroidism (thyroidectomy) were compared with those of acute carbachol pretreatment on the responses of isolated rat atria to the selective beta and alpha adrenoceptor agonists isoproterenol and methoxamine. Both hypothyroidism and acute carbachol pretreatment (3 X 10(-7) -10(-6) M): 1) reduced basal right atrial rates and left atrial tensions; 2) caused an apparent decrease in the inotropic and chronotropic potencies of isoproterenol; 3) reduced the degree of antagonism by propranolol of the responses to isoproterenol; 4) increased the maximum inotropic response of left atria to methoxamine; and 5) converted a lack of response to a positive chronotropic response of right atria to methoxamine. Equivalent reductions of basal rates by hypothermia, or of basal tensions by lowered calcium ion concentrations, did not affect the responses to isoproterenol or methoxamine. The results suggest that both carbachol pretreatment and hypothyroidism functionally antagonize the responses to isoproterenol and enhance the responses to methoxamine by means other than adrenoceptor interconversion.     

Prostaglandin E2 inhibition of secretagogue-stimulated [14C]aminopyrine accumulation in rat parietal cells: a model for its mechanism of action

Prostaglandin E2 (PGE2) differentially inhibited histamine and isoproterenol stimulation of [14C]aminopyrine accumulation in rat parietal cell preparations. Low concentrations of PGE2 decreased the maximum response to isoproterenol whereas higher concentrations increased the EC50 of histamine with only a modest effect on the maximum response. Also, PGE2 potentiated dibutyryl cyclic AMP stimulation of aminopyrine accumulation in either the absence or presence of carbachol. In contrast, PGE2 inhibited potentiation between carbachol and histamine due to its inhibitory effect on histamine and possibly also to an inhibitory effect on cholinergic activity. Islet activating protein prevented the inhibitory actions of PGE2. To account for these results a model is presented based on the recent proposal by Gilman (Cell 36: 577-579, 1984) of an interaction between components of adenylyl cyclase stimulatory and inhibitory guanine nucleotide binding proteins.     

On the use of functional antagonism to estimate dissociation constants for beta adrenergic receptor agonists in isolated guinea-pig trachea.

In guinea-pig trachea, the maximum degree of relaxation that can be elicited by beta adrenergic receptor agonists depends upon the degree of contraction of the smooth muscle induced by cholinergic agonists. In these studies, it is shown that increasing concentrations of carbamylcholine (carbachol) result in a shift to the right of the dose-response curves to (-)-isoproterenol and (-)-soterenol and a reduction of the maximum degree of relaxation produced by these agonists relative to that produced by papaverine. Soterenol is demonstrated to be a partial agonist relative to isoproterenol since its maximum response is reduced to a greater extent by carbachol and it displaces the carbachol dose-response curves to the right less than does isoproterenol. The data were used to calculate, by three different theoretical models of drug-receptor interactions, a dissociation constant (KA) for soterenol. All values obtained were within 2-fold differences. The range of KA values for soterenol was from 5.4 to 9.6 times 10(-8) M. These values are about 100 times larger than the ED50 value for soterenol obtained in the absence of carbachol. The KA value estimated for (-)-isoproterenol (about 3 times 10(-8) M) by one of the models is also around 100 times larger than its ED50 value. This demonstrates further that ED50 values are unreliable indicators of the affinities of agonists for receptors.     

Pharmacologic comparison of selected agonists for the M1 muscarinic receptor in transfected murine fibroblast cells (B82).

The radioligand binding and functional properties of 10 muscarinic agonists for the M1 muscarinic receptors were characterized on the murine fibroblast B82 cells, which have been transfected with the m1 gene. All of the muscarinic agonists completely inhibited [3H](-)methyl-3-quinuclidinyl benzilate binding to the M1 muscarinic receptor in the transfected B82 cells. Their apparent inhibition constant values for agonist/[3H](-)methyl-3-quinuclidinyl benzilate inhibition experiments correlate well with their EC50 values in stimulating phosphatidylinositide hydrolysis. Based on the maximal functional effects: (+)-cismethyl-dioxolane, oxotremorine-M, acetylcholine, carbachol and methacholine are most efficacious, McN-A-343 and arecoline are least efficacious, whereas the efficacies of oxotremorine and pilocarpine are intermediate. In addition, McN-A-343 inhibited carbachol-stimulated phosphatidylinositide hydrolysis. Spare receptors were detected for oxotremorine-M, methacholine and carbachol, but not the rest of the agonists, by comparing the receptor-occupancy curves with the concentration-response curves. These results suggest that the presence of a quaternary nitrogen (trimethylammonium group) within the structure of the agonist may be important for the expression of full agonist activity.     

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)     

Chemical oxidant potentiates electrically and acetylcholine-induced contraction in rat trachea: possible involvement of cholinesterase inhibition

To determine the roles of oxidants in airway responsiveness, we studied the effects of the chemical oxidant N-chlorosuccinimide (NCS) on the contractile responses to electrical field stimulation (EFS) and acetylcholine (ACh) in isolated rat tracheal smooth muscle segments. Effects of NCS on the contractile response to EFS (5 Hz, 20 sec of duration, 50 V) reached the maximum with a 60-min incubation time. NCS potentiated the contractile response to EFS, with a maximum effect at 3 x 10(-7) M and to ACh, with a maximum effect at 3 x 10(-6) M. Thus, at a concentration of 3 x 10(-6) M, NCS significantly decreased log ED50 concentration of ACh from a control value of -5.56 +/- 0.05 to -6.24 +/- 0.06. Physostigmine (10(-7) M), at a concentration that did not alter resting tension, mimicked NCS-induced effects on contractile responses to ACh and EFS with the greater degree of shift in the respective dose-response curves. However, NCS failed to alter dose-response curves to carbachol. Removal of the epithelium shifted the dose-response curves to ACh to lower concentrations, but NCS showed similar effects on dose-response curves to ACh with and without the epithelium. Active staining showed that both acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) activities were found in the smooth muscle of the rat trachea. NCS inhibited both enzyme activities from rat tracheal homogenates in a concentration-dependent fashion. These results suggest that NCS potentiates cholinergically induced contraction by decreasing cholinesterase activity and that the oxidation of cholinesterase may cause hyperresponsiveness of airway smooth muscle by inhibition of the enzyme activity.     

Arachidonic acid metabolites and airway epithelium-dependent relaxant factor

Exogenous arachidonic acid (10(-8) to 10(-4) M) contracted epithelium-free guinea pig tracheal strips. Intact tracheal strips were contracted slightly by low concentrations of arachidonic acid (10(-8) to 10(-5) M), but higher concentrations relaxed them. In contrast, when tracheal strips were precontracted with histamine or carbachol, exogenous arachidonic acid had no effect on epithelium-free preparations but induced concentration-dependent (10(-8) to 10(-4) M) relaxation of intact tracheal strips. The effects of arachidonic acid both in epithelium-free and epithelium-containing trachea were blocked by either indomethacin (10(-6) M) or aspirin (10(-4) M). Studies on the effects of exogenous arachidonic acid, performed with a "sandwich protocol," demonstrated that the postulated airway epithelium-dependent relaxant factor released by an intact tracheal strip relaxes an adjacent epithelium-free strip in the same organ bath. This relaxation is antagonized by indomethacin suggesting the involvement of a cyclooxygenase product in this phenomenon. Comparison of concentration-response curves for contractile agonists in epithelium-free preparations and in one containing epithelium suggests the mobilization of airway epithelium-dependent relaxant factor by histamine but not by carbachol. The effects of cyclooxygenase and lipoxygenase inhibitors indicated that both relaxant and contractile arachidonic acid metabolites are generated by epithelial and nonepithelial cells alike in response to contractile agonists.     

Anti-muscarinic effect of alinidine on acetylcholine-induced vasodilation in isolated and perfused dog coronary arteries.

The effect of alinidine, a bradycardic agent, on the vasodilator responses to acetylcholine was examined in isolated and perfused dog coronary arteries. Single injections of acetylcholine (10(-12)-10(-6) mol) and carbachol (10(-10)-10(-6) mol) produced dose-dependent vasodilations. The endothelial removal by a bolus injection of saponin (1 mg) inhibited those vasodilations. Alinidine (10(-6) M) shifted the dose-response curves of acetylcholine and carbachol to the right, but it did not affect those for isosorbide dinitrate, isoproterenol and adenosine. The rank order of potency of muscarinic antagonists for inhibiting the acetylcholine-induced vasodilation was 4-DAMP greater than or equal to atropine greater than AF-DX 116 greater than or equal to pirenzepine greater than alinidine. Alinidine was approximately 100 times less potent than atropine. Single injection of alinidine (10(-8)-10(-6) mol) dilated the dog coronary artery in a dose-related manner. The vasodilation was not affected by the pretreatment with phentolamine (10(-6) M), pindolol (10(-6) M), atropine (10(-6) M), chlorpheniramine (10(-6) M), cimetidine (10(-6) M) or methysergide (10(-6) M). These results suggest that alinidine has a weak anti-muscarinic effect on the endothelium-dependent vasodilation of the dog coronary artery.     

Biphasic release of endothelium-derived relaxing factor(s) by acetylcholine from perfused canine femoral arteries. Characterization of muscarinic receptors

Experiments were designed to compare the release of endothelium-derived relaxing factor(s) in response to various muscarinic receptor agonists from canine femoral arteries mounted in organ chambers or perfused in a bioassay system. In rings of femoral arteries, suspended for isometric tension recording in organ chambers, acetylcholine induced endothelium-dependent relaxations during contractions evoked by prostaglandin F2 alpha. Atropine and pirenzepine antagonized these relaxations in a competitive manner, atropine with a higher affinity (KB = 1.9 X 10(-9) M) than pirenzepine (KB = 5.4 X 10(-7) M). Carbachol and McN-A-343 also evoked endothelium-dependent relaxations, and pirenzepine inhibited these responses with a similar low potency. The order of relative potency of the agonists in organ chamber studies was acetylcholine = carbachol much greater than McN-A-343. Isolated segments of femoral arteries with endothelium were perfused (2 ml/min) with modified Krebs-Ringer-bicarbonate solution containing indomethacin; the perfusate was bioassayed for endothelium-derived relaxing factor(s) by means of a ring of coronary artery without endothelium. When infused above but not below the femoral artery, low concentrations (10(-8)-10(-7) M) of acetylcholine caused transient relaxations of the bioassay ring contracted with prostaglandin F2 alpha; higher concentrations of acetylcholine caused sustained decreases in tension. Atropine inhibited the two phases of the concentration-relaxation curve with similar potencies. Pirenzepine inhibited both phases in a competitive manner but exhibited significantly higher potency against the first- (ED50, 1.9 X 10(-9) M) than against the second-phase responses (ED50, 2.1 X 10(-7) M). Compound McN-A-343 induced only transient decreases in tension, whereas carbachol caused sustained relaxations.(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.     

Relative potencies of agonists and differential sensitivity to N-ethylmaleimide on muscarinic autoreceptors and postsynaptic receptors in rat hippocampus

Relative potencies of nine muscarinic agonists as activators of autoreceptors regulating [3H]acetylcholine release or of postsynaptic receptors, stimulating inositol monophosphate (IP1) formation were determined in rat hippocampal slices. The agonists could be divided into three groups: 1) full agonists at both sites included oxotremorine-M, carbachol and methacholine; 2) full agonists at autoreceptors and partial agonists at receptors coupled to IP1 formation included oxotremorine, arecoline, bethanechol and RS-86; 3) McN-A-343 was a partial agonist at both sites. Arecaidine propargyl ester was a full agonist at autoreceptors but produced a biphasic stimulation of IP1 formation. Comparison of the EC50 values showed that agonists of groups 1 and 3 were more potent at autoreceptors than at IP1-coupled receptors. Group 2 agonists displayed similar potencies at the two types of receptors. N-Ethylmaleimide (NEM) was more active in antagonizing autoreceptors than IP1-coupled receptors in rat hippocampus. Concentration-response curves to carbachol at autoreceptors were shifted to the right in the presence of 10 microM NEM; 30 microM NEM reduced the maximal response. At postsynaptic receptors, higher concentrations of NEM (100 and 120 microM) were required for inhibition of maximum stimulation of IP1 by carbachol. NEM at 160 and 300 microM abolished the stimulation of IP1 induced by carbachol. These observations provide additional evidence that muscarinic autoreceptors differ from the postsynaptic muscarinic receptors that modulate IP1 formation. The similar rank order of agonist potencies between hippocampal autoreceptors and cardiac muscarinic receptors supports the hypothesis that these autoreceptors are of the M2 (cardiac) subtype.     

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.     

Prostaglandin E2 and [14C]arachidonic acid release by carbachol in the isolated canine parietal cell

Parietal cells have the capacity to synthesize prostaglandins (PGs) and these PGs have a significant effect on parietal cell acid secretion. We examined the stimuli responsible for the control of PG production by parietal cells. Two approaches were utilized. One consisted of measuring PGE2 concentration in the incubation media containing dispersed canine parietal cells stimulated with increasing concentrations of carbachol, histamine plus a phosphodiesterase inhibitor and pentagastrin. PGE2 was measured by radioimmunoassay. The other consisted of measuring the release of radioactive arachidonic acid by parietal cells prelabeled with [4C]arachidonic acid to increasing concentrations of the secretagogues. Both techniques gave very similar results. Only carbachol stimulated the release of PGE2 as well as [14C]arachidonic acid into the incubation media. The increase in PGE2 release was from a base line of 44.3 +/- 10.8 pg/ml to 51.0 +/- 11.7, 55.2 +/- 11.1 and 69.3 +/- 14.3 pg/ml at carbachol concentrations of 10(-6), 10(-5) and 10(-4) M, respectively. In prelabeled cells, carbachol stimulated 1072 +/- 141 and 1264 +/- 155 cpm/ml above basal at concentrations of 10(-5) and 10(-4) M, respectively. Neither histamine nor pentagastrin stimulated PGE2 or [14C]arachidonic acid release significantly at any concentration. The effect of carbachol on the release of [14C]arachidonic acid was blocked by atropine and the exclusion of calcium from the incubation media. Our data suggest that the cholinergic tone to the stomach with subsequent interaction of acetylcholine with muscarinic receptors determines the amount of PG production by the parietal cells. Prostaglandins may then modulate acid secretion by limiting the combined, potentiated effects of the secretagogues.     

Highly potent beta-2 sympathomimetics convert to less potent partial agonists as relaxants of guinea pig tracheae maximally contracted by carbachol. Comparison of relaxation with receptor binding and adenylate cyclase stimulation.

Beta-adrenergic agonists are among to the most potent dilators of airway smooth muscle available and act as functional antagonists of a variety of contractile stimuli. In order to elucidate their loss of relaxant potency in dependence on antagonistic stimuli tracheal relaxation by the beta-2 sympathomimetics (+/-)-salbutamol (+/-)-fenoterol, and (+/-)-formoterol was compared to (-)-isoprenaline in guinea pig tracheae partially and maximally precontracted by carbachol. In partially precontracted tracheae, salbutamol, fenoterol and formoterol exerted maximum relaxation with low EC50 of 20, 5.6 and 0.29 nmol/l, respectively. In maximally precontracted tracheae, however, salbutamol, fenoterol and formoterol were only partial agonists for relaxation with different intrinsic activities (0.62, 0.62 and 0.77, respectively) and increased EC50 (120, 50 and 3.6 nmol/l, respectively). A reduction of relaxant potency by increased muscarinic stimuli was also observed for beta-1 adrenoceptors stimulated by (-)-noradrenaline after blockade of beta-2 adrenoceptors. In order to investigate if the reduced relaxant potency of beta-2 sympathomimetics was caused by a reduced spare receptor capacity or a reduced intrinsic activity for stimulation of adenylate cyclase (AC), we performed experiments in membranes from lung and tracheal cells. In radioligand binding, beta-2 sympathomimetics recognized the high-affinity state (57%) of the beta-2 adrenoceptor with a lower effectiveness than (-)-isoprenaline, which exhibited a 100-fold higher affinity for high over low-affinity states. Dissociation constants for the low-affinity state matched EC50 for AC stimulation. Intrinsic activities (%) for AC stimulation were significantly lower for salbutamol (67%), fenoterol (67%) and formoterol (89%) than for (-)-isoprenaline (100%), indicating that the reduced relaxation potency of the beta-2 sympathomimetics of maximally precontracted tracheae is caused by a reduced intrinsic activity for AC stimulation. It might be speculated that formoterol could improve drug therapy of asthma due to its high binding affinity and its high intrinsic activity for relaxation.     

Osmotic regulation of airway reactivity by epithelium

Inhalation of nonisotonic solutions can elicit pulmonary obstruction in asthmatic airways. We evaluated the hypothesis that the respiratory epithelium is involved in responses of the airways to nonisotonic solutions using the guinea pig isolated, perfused trachea preparation to restrict applied agents to the mucosal (intraluminal) or serosal (extraluminal) surface of the airway. In methacholine-contracted tracheae, intraluminally applied NaCl or KCl equipotently caused relaxation that was unaffected by the cyclo-oxygenase inhibitor, indomethacin, but was attenuated by removal of the epithelium and Na+ and Cl- channel blockers. Na+-K+-2Cl- cotransporter and nitric oxide synthase blockers caused a slight inhibition of relaxation, whereas Na+,K+-pump inhibition produced a small potentiation. Intraluminal hyperosmolar KCl and NaCl inhibited contractions in response to intra- or extraluminally applied methacholine, as well as neurogenic cholinergic contractions elicited with electric field stimulation (+/- indomethacin). Extraluminally applied NaCl and KCl elicited epithelium-dependent relaxation (which for KCl was followed by contraction). In contrast to the effects of hyperosmolarity, intraluminal hypo-osmolarity caused papaverine-inhibitable contractions (+/- epithelium). These findings suggest that the epithelium is an osmotic sensor which, through the release of epithelium-derived relaxing factor, can regulate airway diameter by modulating smooth muscle responsiveness and excitatory neurotransmission.     

Dual effects of dynorphin-(1-13) on cholinergic and substance P-ergic transmissions in the rabbit iris sphincter muscle

In the rabbit iris sphincter muscle, transmural electrical stimulation produces cholinergic and substance P-ergic responses. In the present work, the effects of dynorphin-(1-13), an endogenous ligand of the kappa opioid receptor, on these two neurogenic responses were examined pharmacologically and the data compared to findings in case of other opioid agonists. Dynorphin-(1-13) (10(-7) to 10(-6) M) enhanced the cholinergic responses and attenuated the substance P-ergic response, in a concentration-dependent manner, and these actions of dynorphin-(1-13) were more apparent in the case of low-frequency stimulation. These effects of dynorphin-(1-13) were antagonized by naloxone (10(-5) M). Dynorphin-(1-13) had no effects on the responses to exogenously applied acetylcholine, carbachol and substance P. The augmenting effect on the cholinergic transmission was unique in kappa agonists, as the cholinergic responses were also augmented by other kappa agonists such as dynorphin-(1-17) and ethylketocyclazocine, but attenuated by other opioid agonists (Met-enkephalin, beta-endorphin and morphine) and not affected by SKF-10,047 and nalorphine. On the other hand, the substance P-ergic response was attenuated by all the opioids used. These results suggest that dynorphin-(1-13) presynaptically increases the release of acetylcholine from the parasympathetic postganglionic nerves and reduces the release of substance P from the trigeminal nerve, mediated by kappa type of opioid receptors.     

Isoproterenol-induced inhibition of immunoglobulin E-mediated release of histamine and arachidonic acid metabolites from the human lung mast cell

The inhibitory effect of isoproterenol was examined on the release of histamine, leukotriene C4 (LTC4), and prostaglandin D2 (PGD2) from human lung mast cells. Isoproterenol was more potent in inhibiting anti-immunoglobulin (Ig) E-induced LTC4 and PGD2 release than histamine release from the dispersed lung cell preparations (2-5% mast cells). The negative log molar EC50 values of isoproterenol for inhibiting histamine, LTC4 and PGD2 release were 8.2 +/- 0.2, 8.9 +/- 0.2 and 8.7 +/- 0.3, respectively (mean +/- S.E.M., n = 8). Isoproterenol seldom inhibited histamine release by more than 60%, but usually abolished the release of LTC4 and PGD2. The potency of propranolol (KB = 6 X 10(-10) M) was the same for competitively antagonizing isoproterenol mediated inhibition of histamine, LTC4 or PGD2 release. Using the irreversible beta adrenergic receptor antagonist, bromoacetylalprenololmenthane, the estimated dissociation constant of isoproterenol was 2 X 10(-7) M irrespective of whether inhibition of anti-IgE-induced histamine or LTC4 release was examined. When the divalent cation ionophore A23187 was used to induce histamine release, isoproterenol had either no effect or potentiated the release. In contrast, isoproterenol was capable of virtually abolishing A23187-induced LTC4 release. Qualitatively similar effects of isoproterenol were observed on purified mast cell preparations (greater than 80% mast cells); however, the potency of isoproterenol in inhibiting mediator release from these preparations was reduced. The results demonstrate that there is a substantial receptor reserve for isoproterenol-mediated inhibition of histamine, LTC4 and PGD2 release from the human lung mast cell. The intrinsic efficacy of isoproterenol in inhibiting anti-IgE-induced release of arachidonic acid metabolites is approximately 3 times greater than that for inhibiting histamine release.(ABSTRACT TRUNCATED AT 250 WORDS)