Muscarinic Agonist-Mediated Heterologous Desensitization in Isolated Ileum Requires Activation of Both Muscarinic M2 and M3 Receptors

We investigated the subtypes of the muscarinic receptor mediating short-term heterologous desensitization in the isolated ileum. Treatment of the ileum from C57BL/6 mice with acetylcholine (30 μM) for 20 min caused a subsequent decrease in contractile sensitivity to both prostaglandin F2α (PGF2α) and the muscarinic agonist, oxotremorine-M. This subsensitivity was characterized by 7- and 3-fold increases in the EC50 values of the agonists, respectively, with no significant effect on the maximal response. The subsensitivity to PGF2α was prevented in both M2 and M3 muscarinic receptor knockout mice. Similarly, the subsensitivity to oxotremorine-M was prevented in M2 knockout mice. Acetylcholine-mediated desensitization of histamine-induced contractions in the guinea pig ileum was inhibited by both M2- and M3-selective muscarinic antagonists with high potency, although careful analysis of the data suggested behavior more consistent with an M2 antagonistic profile. Modeling studies showed that the competitive antagonism of response contingent upon activation of two receptor subtypes should exhibit a pharmacological profile similar to that of the least sensitive signaling pathway. Our results demonstrate that muscarinic agonist-mediated short-term heterologous desensitization of intestinal smooth muscle is contingent upon activation of both M2 and M3 muscarinic receptors and that activation of either receptor by itself is insufficient to cause desensitization

Activation of N,N-Dimethylaminoazobenzene catalyzed by peroxidase

The mechanism for the metabolic activatiou of N,N-dimethylaminoazobenzene is unknown. Although cytochrome P-450 dependent mixed function oxygenases are important in the activation of arylamines, there are target tissues for arylamines which do not contain these oxygenases. These tissues do contain peroxidases. Therefore a one electron oxidation mechanism was investigated by studying a Horseradish Peroxidase/hydrogen peroxide catalyzed metabolism of aminoazobenzenes [i.e; N-methyl-4-aminoazobenzene (MAB), N,N-dimethyl-4-aminoazobenzene (DAB), aminoazobenzene (AB)], and by following the binding of the activated products to exogenous DNA. In order to further explore the mechanism, the effect of biological and phenolic antioxidants on metabolism and binding of the activated products of [¹⁴C] MAB was studied. The oxidation products were analyzed by spectrophotometry, high pressure liquid chromatography and thin layer chromatography. The major product obtained from both MAB and DAB at pH 7.4 was a dimer of MAB, i.e; N-methyl,N(MAB)-4-aminoazobenzene. The product obtained at pH 5.0 was 4'-(MAB)-N-methyl-4-aminoazobenzene. It is suggested that DAB is first N-demethylaced, and then follows the same metabolic pathway as does MAB. Evidence suggests that AB is also oxidized to a dimer with N-N linkage at pH 7.4 and C-N linkage at pH 5.0. -- The binding of the activated products to calf thymus DNA was observed spectrophotometrically. [¹⁴C] MAB was used to determine the quantitative binding by liquid scintillation counting. DNA is attacked by a free radical of MAB, and also by other radicals formed in chain propagating reactions with the MAB radical. The maximum binding of [¹⁴C] MAB to homo polyriboguanylic acid among the four homo polyribonucleotides suggests the preferential binding of the activated products occurs at the guanine residues in DNA. -- The phenolic and biological antioxidants inhibit the DNA binding either by inhibiting the total oxidation of MAB or by converting the reactive metabolites to detoxication products

L-ornithine derived polyamines in cystic fibrosis airways.

Increased arginase activity contributes to airway nitric oxide (NO) deficiency in cystic fibrosis (CF). Whether down-stream products of arginase activity contribute to CF lung disease is currently unknown. The objective of this study was to test whether L-ornithine derived polyamines are present in CF airways and contribute to airway pathophysiology. Polyamine concentrations were measured in sputum of patients with CF and in healthy controls, using liquid chromatography-tandem mass spectrometry. The effect of spermine on airway smooth muscle mechanical properties was assessed in bronchial segments of murine airways, using a wire myograph. Sputum polyamine concentrations in stable CF patients were similar to healthy controls for putrescine and spermidine but significantly higher for spermine. Pulmonary exacerbations were associated with an increase in sputum and spermine levels. Treatment for pulmonary exacerbations resulted in decreases in arginase activity, L-ornithine and spermine concentrations in sputum. The changes in sputum spermine with treatment correlated significantly with changes in L-ornithine but not with sputum inflammatory markers. Incubation of mouse bronchi with spermine resulted in an increase in acetylcholine-induced force and significantly reduced nitric oxide-induced bronchial relaxation. The polyamine spermine is increased in CF airways. Spermine contributes to airways obstruction by reducing the NO-mediated smooth muscle relaxation

Acetylcholine-Induced Desensitization of Muscarinic Contractile Response in Guinea Pig Ileum Is Inhibited by Pertussis Toxin Treatment

We investigated the effects of pertussis toxin treatment on acetylcholine-induced desensitization of the muscarinic contractile response in guinea pig ileum. Incubation of the isolated ileum with acetylcholine (30 μM) for 20 min caused a decrease in the sensitivity of the ileum to the contractile action of the muscarinic agonist oxotremorine-M. This desensitization was characterized by an increase in the EC50 value of oxotremorine-M without a change in its maximal effect. A maximal 4- to 5-fold increase in the EC50value of oxotremorine-M was measured at the earliest time investigated after acetylcholine treatment (5 min), and normal sensitivity recovered within approximately 20 min after washout of acetylcholine. Treatment of the ileum with pertussis toxin caused a small increase in the contractile response to oxotremorine-M when measured without prior exposure to acetylcholine. After exposure to acetylcholine, little desensitization was observed in ilea that had been treated with pertussis toxin. Pertussis toxin-treatment caused a small increase in oxotremorine-M-mediated phosphoinositide hydrolysis and a large decrease in oxotremorine-M-mediated inhibition of forskolin-stimulated cAMP accumulation in slices of the longitudinal muscle of the ileum. Exposure of the ileum to acetylcholine had no desensitizing effect on the ability of oxotremorine-M to elicit phosphoinositide hydrolysis, indicating that the mechanism for desensitization of the contractile response occurs at a level downstream from the receptor and phosphoinositide hydrolysis. Our results suggest that activation of muscarinic receptors coupled to pertussis toxin-sensitive Gi and Go is required for most of the desensitization observed in this study

Increased Relaxant Action of Forskolin and Isoproterenol against Muscarinic Agonist-Induced Contractions in Smooth Muscle from M2 Receptor Knockout Mice

The ability of forskolin and isoproterenol to inhibit the contractile action of the muscarinic agonist, oxotremorine-M, was investigated in smooth muscle from wild-type and M2 muscarinic receptor knockout mice. Forskolin (5.0 μM) caused a significant reduction in the contractile activity of oxotremorine-M in ileum, trachea, and urinary bladder from both wild-type and M2 muscarinic receptor knockout mice. This reduction in contractile activity was characterized by decreases in potency or maximal response, but not always both. Similar results were obtained with isoproterenol (1.0 μM). The relaxant effects of forskolin in ileum, trachea, and urinary bladder from M2 receptor knockout mice were approximately 3- to 9-fold greater than those observed in the same tissues from wild-type mice. Similar results were obtained with isoproterenol in ileum and urinary bladder, although the differences between wild-type and M2 receptor knockout tissues were less than those observed with forskolin. In contrast, there was no significant difference between the relaxant effect of isoproterenol in trachea from wild-type and M2 receptor knockout mice. In contrast to the results observed with oxotremorine-M as the contractile agent, forskolin and isoproterenol did not exhibit greater relaxant activity against KCl-induced contractions in M2 receptor knockout mice compared with wild-type mice. These results suggest that a component of the contractile response to muscarinic agonists in smooth muscle involves an M2 muscarinic receptor-mediated inhibition of the relaxant effects of agents that increase cAMP levels

Comparison of the Antimuscarinic Action of p-Fluorohexahydrosiladifenidol in Ileal and Tracheal Smooth Muscle

We investigated the ability of the muscarinic antagonist p-fluorohexahydrosiladifenidol to inhibit muscarinic agonist-induced contractions and phosphoinositide hydrolysis in the guinea pig ileum and trachea. This antagonist displayed higher potency at blocking oxotremorine-M-induced contractions of the ileum compared with those of the trachea. When estimated using a simple model for competitive antagonism, the observed dissociation constant of p-fluorohexahydrosiladifenidol exhibited approximately 12-fold higher potency in the ileum compared with the trachea. We also investigated the ability of p-fluorohexahydrosiladifenidol to affect the inhibition of contraction caused by the known competitive muscarinic antagonist atropine. Using resultant analysis to analyze this interaction, we found that the true dissociation constant of p-fluorohexahydrosiladifenidol for competitively antagonizing oxotremorine-M-induced contractions in the ileum exhibited significantly lower potency than when calculated assuming a simple competitive model. In contrast, resultant analysis showed little difference between the true and observed potencies of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced contractions in the trachea. Using a simple competitive model, we found little difference in the observed dissociation constant of p-fluorohexahydrosiladifenidol for antagonizing oxotremorine-M-induced phosphoinositide hydrolysis in guinea pig ileum and bovine trachea. We also noted that p-fluorohexahydrosiladifenidol (0.3–1.0 μM) moderately inhibited histamine-induced contractions of ileum but not of trachea. Our results suggest that p-fluorohexahydrosiladifenidol does not discriminate markedly between M3 muscarinic receptors in the ileum and trachea and that it may posses a more potent, nonmuscarinic inhibitory effect on contraction in the ileum

Spermine effect on mice bronchial smooth muscle contractile properties.

<p>A) Bronchial rings stimulated with acethylcholine (10−5 M) in the absence (Control; N = 6 rings) and presence (N = 10 rings) of spermine (10−4 M) or KCl (128 mM). B) Sodium nitroprusside (SNP) dose-response relaxation curves of acetylcholine pre-contracted (E75 = 10−5 M) bronchial smooth muscle in the absence (Control; N = 6 rings) and presence (N = 10 rings) of spermine (10−4 M). A typical SNP-induced relaxation tracing in the absence (left panel) and presence (right panel) of spermine is shown. Following ACH Stimulation (open triangle) the sustained bronchial muscle force in the presence of spermine is greater than in controls. * P<0.01 versus control by unpaired Student t-test (A) or two-way ANOVA with Tukey-Kramer multiple comparison testing (B).</p

Demographics of study populations.

<p>Gender: F = females, M = males. Age: numbers represent mean (range) age in years. FEV₁: values represent FEV₁ in % of predicted vales (24).</p