Diverse developing mouse lineages exhibit high-level c-Myb expression in immature cells and loss of expression upon differentiation.
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The c-myb gene encodes a sequence specific transactivator that is required for fetal hematopoiesis, but its potential role in other tissues is less clear because of the early fetal demise of mice with targeted deletions of the c-myb gene and incomplete of knowledge about c-myb's expression pattern. In the hematopoietic system, c-Myb protein acts on target genes whose expression is restricted to individual lineages, despite Myb's presence and role in multiple immature lineages. This suggests that c-Myb actions within different cell type-specific contexts are strongly affected by combinatorial interactions. To consider the possibility of similar c-Myb actions could extend into non-hematopoietic systems in other cell and tissue compartments, we characterized c-myb expression in developing and adult mice using in situ hybridization and correlated this with stage-specific differentiation and mitotic activity. Diverse tissues exhibited strong c-myb expression during development, notably tooth buds, the thyroid primordium, developing trachea and proximal branching airway epithelium, hair follicles, hematopoietic cells, and gastrointestinal crypt epithelial cells. The latter three of these all maintained high expression into adulthood, but with characteristic restriction to immature cell lineages prior to their terminal differentiation. In all sites, during fetal and adult stages, loss of c-Myb expression correlated strikingly with the initiation of terminal differentiation, but not the loss of mitotic activity. Based on these data, we hypothesize that c-Myb's function during cellular differentiation is both an activator of immature gene expression and a suppressor of terminal differentiation in diverse lineages. (+info)
Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells.
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To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (alpha, beta and gamma)-selective agonists and RARalpha- and RARgamma-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARalpha- and RARgamma-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARbeta-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 microM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARalpha antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARalpha- and RARgamma-selective retinoids. In contrast, the RARgamma antagonist only weakly inhibited RARalpha-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARgamma-selective retinoid. Our studies indicate that RARalpha is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARgamma isotype can also induce mucin genes. Furthermore these studies suggest that RARbeta is probably not (directly) involved in RA-induced mucin gene expression. (+info)
Comparison of functional antagonism between isoproterenol and M2 muscarinic receptors in guinea pig ileum and trachea.
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The ability of the M2 muscarinic receptor to mediate an inhibition of the relaxant effects of forskolin and isoproterenol was investigated in guinea pig ileum and trachea. In some experiments, trachea was first treated with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) mustard to inactivate M3 receptors. The contractile response to oxotremorine-M was measured subsequently in the presence of both histamine (10 microM) and isoproterenol (10 nM). Under these conditions, [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3b]-[1,4]benzodiazepine-6-one (AF-DX 116) antagonized the contractile response to oxotremorine-M in a manner consistent with an M3 mechanism. However, when the same experiment was repeated using forskolin (4 microM) instead of isoproterenol, the response to oxotremorine-M exhibited greater potency and was antagonized by AF-DX 116 in a manner consistent with an M2 mechanism. We also measured the effects of pertussis toxin treatment on the ability of isoproterenol to inhibit the contraction elicited by a single concentration of either histamine (0.3 microM) or oxotremorine-M (40 nM) in both the ileum and trachea. Pertussis toxin treatment had no significant effect on the potency of isoproterenol for inhibiting histamine-induced contractions in the ileum and trachea. In contrast, pertussis toxin treatment enhanced the relaxant potency of isoproterenol against oxotremorine-M-induced contractions in the ileum but not in the trachea. Also, pertussis toxin treatment enhanced the relaxant potency of forskolin against oxotremorine-M-induced contractions in the ileum and trachea. We investigated the relaxant potency of isoproterenol when very low, equi-effective (i.e., 20-34% of maximal response) concentrations of either histamine or oxotremorine-M were used to elicit contraction. Under these conditions, isoproterenol exhibited greater relaxant potency against histamine in the ileum but exhibited similar relaxant potencies against histamine and oxotremorine-M in the trachea. Following 4-DAMP mustard treatment, a low concentration of oxotremorine-M (10 nM) had no contractile effect in either the ileum or trachea. Nevertheless, in 4-DAMP mustard-treated tissue, oxotremorine-M (10 nM) reduced the relaxant potency of isoproterenol against histamine-induced contractions in the ileum, but not in the trachea. We conclude that in the trachea the M2 receptor mediates an inhibition of the relaxant effects of forskolin, but not isoproterenol, and the decreased relaxant potency of isoproterenol against contractions elicited by a muscarinic agonist relative to histamine is not due to activation of M2 receptors but rather to the greater contractile stimulus mediated by the M3 receptor compared with the H1 histamine receptor. (+info)
Kinetic analysis of drug-receptor interactions of long-acting beta2 sympathomimetics in isolated receptor membranes: evidence against prolonged effects of salmeterol and formoterol on receptor-coupled adenylyl cyclase.
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The long-acting beta2 sympathomimetics salmeterol and formoterol have been presumed to exert their prolonged action either by binding to an accessory binding site ("exo-site") near the beta2 adrenoceptor or by their high affinity for beta2 adrenoceptors and correspondingly slow dissociation. Whereas most studies with salmeterol had been done in intact tissues, which have slow diffusion and compartmentation of drugs in lipophilic phases, that restrict drug access to the receptor biophase, we used purified receptor membranes from rat lung and disaggregated calf tracheal myocytes as model systems. Binding experiments were designed to measure the slow dissociation of agonists by means of delayed association of (-)-[125I]iodopindolol. Rat lung membranes were pretreated with high concentrations of agonists (salmeterol, formoterol, isoprenaline) before dissociation was induced by 50-fold dilution. Half-times of association of (-)-[125I]iodopindolol remained unchanged compared with untreated controls, indicating that dissociation of agonists occurred in less than 2 min. Adenylyl cyclase experiments were designed to determine the on and off kinetics of agonists to beta2 adrenoceptors by measuring the rate of receptor-induced cyclic AMP (cAMP) formation. Experiments were performed in tracheal membranes characterized by high Vmax values of cAMP formation. Adenylyl cyclase activation occurred simultaneously with the addition of the agonist, continued linearly with time for 60 min, and ceased immediately after the antagonist was added. Similarly, when receptor membranes were preincubated in a small volume with high salmeterol concentrations, there was a linear increase in cAMP formation, which was immediately interrupted by a 100-fold dilution of the reaction mixture. This militates against the exo-site hypothesis. On the other hand, dissociation by dilution was much less when membranes were preincubated with a large volume of salmeterol at the same concentration, indicating that physicochemical effects, and not exo-site binding, underlie its prolonged mode of action. (+info)
The contribution of extraneuronal uptake to the trachea-blood vessel selectivity of beta-adrenoceptor stimulants in vitro in guinea-pigs.
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1 The potencies relative to isoprenaline of isoetharine, tertiary butyl noradrenaline, salbutamol, orciprenaline, Me 506, rimiterol, fenoterol, carbuterol and terbutaline on isolated preparations of guinea-pig trachea and blood vessels (perfused hind limb) were determined. All the compounds were selective for trachea and selectivity values, i.e. relative potency on trachea divided by relative potency on hind limb, ranged from 2.3 to 21.4. 2 Responses to isoprenaline (the reference compound), tertiary butyl noradrenaline and isoetharine were potentiated on trachea by 50 muM phenoxybenzamine (PHB) and by other inhibitors of extraneuronal uptake (ENU). Under these conditions the selectivity values of all the compounds was close to unity. 3 Selectivity values were also close to unity if they were calculated from data obtained without ENU inhibition, provided that only those compounds not potentiated by PHB on trachea were used. 4 It is proposed that the trachea-blood vessel selectivity shown by beta-adrenoceptor stimulants can be caused by the influence of ENU upon them, rather than by their ability to distinguish between two beta2-adrenoceptors. 5 The suggestion that differences exist between beta2-adrenoceptors in respiratory and vascular smooth muscle is not supported by the in vitro experiments described. (+info)
The cat lung strip as an in vitro preparation of peripheral airways: a comparison of beta-adrenoceptor agonists, autacoids and anaphylactic challenge on the lung strip and trachea.
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1 A new in vitro preparation, the isolated lung strip of the cat, is described for investigating the direct effect of drugs on the smooth muscle of the peripheral airways of the lung. The preparation comprises a thin strip of lung parenchyma which can be mounted in a conventional organ bath for isometric tension recording. Its pharmacological responses have been characterized and compared with the isolated tracheal preparation of the cat. 2 The lung strip exhibited an intrinsic tone which was relaxed by catecholamines, aminophylline and flufenamate. It was contracted strongly by histamine, prostaglandin F2alpha, acetylcholine, compound 48/80, potassium depolarizing solution and alternating current field stimulation. In contrast, the cat trachea was unresponsive to histamine and prostaglandin F2alpha and did not exhibit an intrinsic tone. 3 (-)-Isoprenaline and (-)-adrenaline were much more potent in relaxing the lung strip than the trachea. The potency order of relaxation responses to isoprenaline, adrenaline and (+/-)-noradrenaline in the lung strip was isoprenaline greater than adrenaline greater than noradrenaline but in the trachea was isoprenaline greater than noradrenaline greater than or equal to adrenaline. 4 beta2-Adrenoceptor selective agonists salbutamol and terbutaline were more potent in the lung strip than the trachea, suggesting beta2-adrenoceptors predominated in the lung strip. Propranolol was equipotent in inhibiting isoprenaline relexations of the lung strip and trachea, whereas practolol was much less effective in inhibiting lung strip than trachea, further supporting a predominance of beta2-adrenoceptors in lung strip and beta1-adrenoceptors in trachea. 5 Strong Schultz-Dale type contractions were elicited in both lung strips and trachea by Ascaris lumbricoides antigen in actively sensitized cats. The initial phase of the contractile response of the lung strip following challenge was shown to be due to histamine release and was absent in the trachea. The delayed phase of the contraction which took several minutes to develop in both the mepyramine-treated lung strip and trachea was not due to prostaglandins E1, F2alpha or bradykinin, the probable mediator being slow reacting substance of anaphylaxis (SRS-A). 6 It is concluded that the isolated lung strip of the cat is useful as an in vitro model for investigating the effect of drugs on the smooth muscle of the peripheral airways of the lungs. (+info)
Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release.
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1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea. (+info)
Anaphylactic bronchoconstriction in BP2 mice: interactions between serotonin and acetylcholine.
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1. Immunized BP2 mice developed an acute bronchoconstriction in vivo and airway muscle contraction in vitro in response to ovalbumin (OA) and these contractions were dose dependent. 2. Methysergide or atropine inhibited OA-induced bronchoconstriction in vivo and airway muscle contraction in vitro. 3. Neostigmine potentiated the OA-induced bronchoconstriction in vivo and airway muscle contraction in vitro of BP2 mice. This potentiation was markedly reduced by the administration of methysergide or atropine and when the two antagonists were administered together, the responses were completely inhibited. 4. Neostigmine also potentiated the serotonin (5-HT)- and acetylcholine (ACh)-induced bronchoconstriction and this potentiation was significantly reversed by atropine. 5. These results indicate that OA provokes a bronchoconstriction in immunized BP2 mice by stimulating the release of 5-HT, which in turn acts via the cholinergic mediator, ACh. (+info)