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Biomedical subjects

J C Mak

Publications and source records attributed to J C Mak.

At least 37 records · Page 2Linked to original sources
PubMed1997

Glucocorticoids induce beta2-adrenergic receptor function in human nasal mucosa.

Glucocorticoids are hypothesized to induce beta2-adrenergic receptors (beta2-R) and their functions. The ability of dexamethasone (DEX) in vitro and beclomethasone dipropionate (BDP) in vivo to induce beta2-R messenger RNA (mRNA) and function was investigated in human nasal mucosa. In this tissue, albuterol does not stimulate exocytosis either in vivo or in vitro (Mullol and coworkers, 1992). Therefore, induction of beta2-R-mediated glandular exocytosis by glucocorticoids was proposed as an unambiguous outcome measure. Human nasal mucosa was cultured for 3 d with and without 1 microM DEX, then challenged with media or 100 microM albuterol. Culture supernatants were collected for measurement of exocytosed glandular products. Explant mRNA was extracted for reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization of beta2-R mRNA performed. In vivo, normal subjects received saline or BDP for 3 d before albuterol nasal provocation. Concentrations of exocytosed products were measured in nasal secretions. RNA was extracted from nasal epithelial scrapings for RT-PCR. In vitro, DEX treatment induced albuterol-mediated glandular exocytosis (p < 0.04), and increased the steady-state beta2-R/beta-actin mRNA ratio (p < 0.05), and expression of beta2-R mRNA in glands. In vivo, BDP increased the beta2-R/beta-actin mRNA ratio in epithelial scrapings (p < 0.04), but did not induce albuterol-mediated glandular secretion. We conclude that glucocorticoids increase steady-state beta2-R mRNA levels in vivo and in vitro, and can induce beta2-R function as assessed by submucosal gland exocytosis in vitro. While topical BDP induced epithelial beta2-R mRNA, it did not modulate exocytosis from the deeper submucosal glands.

Administration, Topical↗
PubMed1996

Effect of short- and long-acting beta 2-adrenoceptor agonists on pulmonary beta 2-adrenoceptor expression in human lung.

beta-Adrenoceptor agonists induce the down-regulation of beta 2-adrenoceptors and mRNA expression in animal lung. The down-regulation of beta 2-adrenoceptors may limit the therapeutic efficacy of beta 2-adrenoceptor-mediated bronchodilators in obstructive airways disease. We examined the effects of three selective beta 2-adrenoceptor agonists, salbutamol, salmeterol and formoterol on beta 2-adrenoceptor binding and mRNA levels in human lung in vitro. Human lung was obtained from cardiac transplantation donors. Peripheral lung was chopped and incubated with three selective beta 2-adrenoceptor agonist for 3 h or 24 h at three different concentrations (0.1, 1 and 10 microM). The affinity and density of beta 2-adrenoceptors was determined by [125I]iodocyanopindolol equilibrium binding in a lung membrane preparation in the presence of 0.1 microM CGP 20712 A (1-{2-[(3-carbamoyl-4-hydroxy)phenoxy] ethylamino}-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-propan-2-ol), a selective beta 1-adrenoceptor antagonist. Although treatment with salbutamol for 3 h did not change beta 2-adrenoceptor density, both salmeterol and formoterol reduced beta 2-adrenoceptor density, and exposure to each agonist for 24 h reduced beta 2-adrenoceptor density at all concentrations. Treatment with 10 microM salmeterol increased the equilibrium dissociation constant (Kd), and also shifted the competition curves of (-)-isoprenaline to the left, beta 2-Adrenoceptor mRNA, measured by Northern blot analysis using a human beta 2-adrenoceptor cDNA probe, was reduced after exposure to all beta 2-adrenoceptor agonists at 3 h. Our data provide evidence for down-regulation of beta 2-adrenoceptor protein and mRNA after selective beta 2-adrenoceptor agonist treatment in human lung.

Adolescent↗
PubMed1996

Mechanisms of impaired beta-adrenoceptor-induced airway relaxation by interleukin-1beta in vivo in the rat.

We studied the in vivo mechanism of beta-adrenergic receptor (beta-AR) hyporesponsiveness induced by intratracheal instillation of interleukin-1beta (IL-1beta, 500 U) in Brown-Norway rats. Tracheal and bronchial smooth muscle responses were measured under isometric conditions ex vivo. Contractile responses to electrical field stimulation and to carbachol were not altered, but maximal relaxation induced by isoproterenol (10(-6)-10(-5) M) was significantly reduced 24 h after IL-1beta treatment in tracheal tissues and to a lesser extent, in the main bronchi. Radioligand binding using [125I]iodocyanopindolol revealed a 32+/-7% reduction in beta-ARs in lung tissues from IL-1beta-treated rats, without any significant changes in beta2-AR mRNA level measured by Northern blot analysis. Autoradiographic studies also showed significant reduction in beta2-AR in the airways. Isoproterenol-stimulated cyclic AMP accumulation was reduced by IL-1beta at 24 h in trachea and lung tissues. Pertussis toxin reversed this hyporesponsiveness to isoproterenol but not to forskolin in lung tissues. Western blot analysis revealed an IL-1beta-induced increase in Gi(alpha) protein expression. Thus, IL-1beta induces an attenuation of beta-AR-induced airway relaxation through mechanisms involving a reduction in beta-ARs, an increase in Gi(alpha) subunit, and a defect in adenylyl cyclase activity.

Animals↗
PubMed1996

On muscarinic control of neurogenic mucus secretion in ferret trachea.

1. Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35SO4 as a label for secreted mucus, and the muscarinic receptor antagonists telenzepine for the M1 receptor subtype, methoctramine for the M2 subtype and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) for the M3 receptor. We also performed receptor binding and mapping studies. 2. Each muscarinic antagonist displaced [N-methyl-3H]scopolamine binding with high-affinity binding constant (KH) values of 1.9, 2.7 and 5.0 nM for telenzepine, methoctramine and 4-DAMP, respectively. Muscarinic M1 and M3 receptors localized to submucosal glands, whereas M2 receptors did not. 3. In vitro, electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 160%. Telenzepine did not inhibit the neurogenic secretory response at concentrations two-or twentyfold its KH value, nor did it inhibit secretion induced by acetylcholine (ACh). 4-DAMP inhibited neurogenic secretion by 80 and 95%, respectively, at concentrations two-and twentyfold its KH value, and also inhibited ACh-induced secretion. Methoctramine potentiated neurogenic secretion induced at 2.5 Hz (50 V, 0.5 ms for 5 min) in a dose-related (5.4-100 nM) manner with increases of 33-451% above electrically stimulated values. Methoctramine did not potentiate secretion induced at 10 Hz and did not have any effect on ACh-induced secretion. 4. In vivo, vagal stimulation (10 V, 10 Hz, 2 ms for 8 min) increased output of 35SO4 by approximately 120%. Telenzepine had no significant effect on neurogenic secretion. Methoctramine approximately doubled the stimulated response, whereas 4-DAMP abolished the stimulated secretory response. 5. We conclude that in ferret trachea, cholinergic nerve stimulation increases mucus secretion via muscarinic M3 receptors on the submucosal glands. The magnitude of the secretory response is regulated by neuronal M2 muscarinic receptors. The muscarinic M1 receptors localized to the submucosal glands do not appear to be involved with mucus secretion.

Animals↗
PubMed1996

Localisation and expression of beta-adrenoceptor subtype mRNAs in human lung.

The cellular localisation and distribution of mRNAs encoding beta-adrenoceptor subtypes in human lung were studied by in situ hybridisation and Northern blot analysis. The 851-bp SmaI/PvuII fragment of human beta 1-adrenoceptor cDNA, the 439-bp SmaI fragment of human beta 2-adrenoceptor cDNA and the 975-bp SmaI fragment of human beta 3-adrenoceptor cDNA bound to single mRNA species of approximately 3.2 kb, 2.2 kb and 2.3 kb in size, respectively. Human lung and heart and rabbit lung expressed both beta 1- and beta 2-adrenoceptor mRNAs with no detectable level of beta 3-adrenoceptor mRNA, while rabbit perirenal adipose tissue expressed beta 1-, beta 2- and beta 3-adrenoceptor mRNAs. Cultured human airway epithelial cells and airway smooth muscle cells expressed only beta 2-adrenoceptor mRNA. In situ hybridisation in human lung, using 35S-labelled antisense RNA probes revealed a high level of expression of beta 1- and beta 2-adrenoceptor mRNAs in the pulmonary blood vessels, high level of expression of beta 2-adrenoceptor mRNA in the alveolar walls with less expression of beta 1-adrenoceptor mRNA. There was a moderate expression of beta 2-adrenoceptor but not beta 1-adrenoceptor mRNA in airway epithelium and smooth muscle of peripheral airways and no detectable beta 3-adrenoceptor mRNA in any lung structures.

Animals↗
PubMed1996

Autoradiographic mapping of beta-adrenoceptors in human skin.

A high density of beta 2-adrenoceptors has been found in human skin. Using autoradiographic mapping we investigated the distribution of beta 1- and beta 2-receptors in normal and diseased human skin. Cryostat sections of human skin obtained at biopsy were incubated with [125I]-iodocyanopindolol and nonspecific binding was identified by incubation of adjacent sections with 200 microM (-)-isoproterenol; beta 2-receptors were visualized using CGP 20712A and beta 1-receptors using ICI 118,551 as competing agents. The epidermis was densely labelled with an even distribution throughout all layers. Most of the beta-receptors were of the beta 2-subtype, with practically no beta 1-receptors. beta-Receptors were also localized to eccrine sweat glands, dermal blood vessels, and perivascular inflammatory cells, but there was no labelling of sebaceous glands. Topical glucocorticoids caused an increase in the density of epidermal beta-receptors. We conclude that keratinocytes and eccrine sweat glands express high densities of beta 2-receptors, suggesting that they may have a physiological role in the regulation of these cells.

Autoradiography↗
PubMed1996

Autoradiographic mapping of pulmonary NK1 and NK2 tachykinin receptors and changes after repeated antigen challenge in guinea pigs.

An autoradiographic technique was used to study the distribution of changes in pulmonary NK1 and NK2 receptors in guinea pig lung after repeated antigen challenge. Specific labeling of [3H]CP96345 (NK1 receptors) and [3H]SR48968 (NK2 receptors) was localized over the tracheal and bronchial smooth muscle; the density of binding increased towards smaller airways with a higher density for [3H]CP96345 binding. Bronchial epithelium and pulmonary blood vessels were also labeled densely with [3H]CP96345. No remarkable difference in the pattern of distribution of pulmonary NK1 and NK2 tachykinin receptors was observed between control, vehicle-challenged, and repeatedly antigen-challenged (weekly for three times) guinea pigs. A significant reduction in specific labeling of [3H]CP96345 (p < 0.01) and [3H]SR48968 (p < 0.05) over pulmonary structures was observed in antigen-challenged compared to control or vehicle-challenged animals. This study provides evidence that NK1 and NK2 tachykinin receptors are both localized to smooth muscle of all sizes in guinea pig airways and provides further evidence for a discrete distribution of NK1 and NK2 tachykinin receptors, consistent with their relative functional activities. In an established model of airway inflammation a decrease in the expression of NK1 and NK2 tachykinin receptors was evident on several different cell types within the lung, and this could influence airway and vascular reactivity.

Animals↗
PubMed1996

Muscarinic and beta-adrenergic receptor expression in peripheral lung from normal and asthmatic patients.

We used human peripheral lung from 8 mildly asthmatic patients and 11 normal donors to study the expression of muscarinic and beta-adrenergic receptors in asthma. There was no significant difference in the affinity or the density of muscarinic (labeled with [N-methyl-3H]scopolamine) and beta 1- and beta 2-adrenergic receptors (labeled with [125I]iodocyanopindolol) in peripheral lung from asthmatics compared with nonasthmatics. Only the muscarinic m1 receptor mRNA was detected in human lung using Northern blot analysis. Additionally, peripheral lung cellular mRNA hybridized to human beta 1 and beta 2 cDNA probes, giving 3.2- and 2.2-kb hands corresponding to beta 1 and beta 2-adrenergic receptors mRNA, respectively. Densitometric scanning of the autoradiograms suggests that there was no significant difference in the relative abundance of muscarinic m1 and beta 1- and beta 2-adrenergic receptor mRNA in asthmatic compared with nonasthmatic lungs. Functional experiments obtained in trachea suggest that there was an increase in the cholinergic neural response evoked by electrical field stimulation in asthmatic compared with nonasthmatic tissues which was not due to a reduction in inhibitory noncholinergic nonadrenergic relaxations.

Adult↗
PubMed1996

Beta-Adrenoceptor-medicated down-regulation of M2 muscarinic receptors: role of cyclic adenosine 5'-monophosphate-dependent protein kinase and protein kinase C.

Stimulation of beta2-adrenoceptors with the selective beta2 agonist procaterol caused a biphasic decrease in cell surface M2 muscarinic receptor number in human embryonic lung 299 cells when measured with the hydrophilic antagonist [3H]N-methylscopolamine. In contrast, total muscarinic receptor number, measured with the lipophilic antagonist [3H]quinuclidinylbenzilate, decreased after only 24-hr treatments with procaterol. The loss in receptor number at 24 hr was mimicked with the use of forskolin and the cAMP analogue 8-bromo-cAMP, indicating a cAMP-mediated mechanism. Northern blot analysis showed a small and transient increase in m2-receptor mRNA levels up to 2 hr but no long term (24 hr) effect. Chronic (24 hr) treatment with 8-bromo-cAMP also had no effect on m2 muscarinic receptor mRNA, whereas forskolin caused a 50% reduction in the steady state levels of m2 mRNA that could be only partially blocked by the cAMP-dependent protein kinase inhibitor H-8 and the protein kinase C inhibitor GF 109203X. Procaterol-induced down-regulation of M2 receptors was fully blocked by N-[2-(methylamino)ethyl]-5'-isoquinoline-sulfonamide and 2-[1-(3-dimethylaminopropyl)-inol-3-yl]-3-(indol-3-yl)maleimide, implicating both of these kinases in the M2 muscarinic receptor down-regulation. Conversely, the forskolin- and 8-bromo-cAMP-induced down-regulation was only partially inhibited and unaffected by these inhibitors, respectively. In control cells and those treated with procaterol for < / = 2 hr, cAMP generation was significantly inhibited by carbachol. The inhibitory effect of carbachol was, however, lost after 24-hr exposure to procaterol. This desensitization was partially reversed by preincubations with H-8 and GF 109203X. Collectively, these results suggest that transregulation of M2 muscarinic receptors by beta2-adrenoceptor stimulation can be demonstrated at the protein level in human embryonic lung 299 cells. Furthermore, a role is suggested for cAMP-dependent kinase and PKC in M2 muscarinic receptor down-regulation and their functional desensitization.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1996

Transforming growth factor-beta 1 induces transcriptional down-regulation of m2 muscarinic receptor gene expression.

In human embryonic lung fibroblasts, transforming growth factor-beta 1 (TGF-beta 1) induced a time-dependent down-regulation of M2 muscarinic receptor binding sites as measured with the nonselective hydrophilic ligand [3H]N-methylscopolamine (NMS). This down-regulation was slow, with 58% loss of all receptors after 24 hr of treatment. The affinity of [3H]NMS for the remaining sites was unaltered by TGF-beta 1. The loss in [3H]NMS binding was accompanied by reduced adenylyl cyclase activity and functional desensitization of M2 muscarinic receptors. Northern blot analyses showed a 72% decrease in the steady state levels of m2 muscarinic receptor mRNA after 24-hr TGF-beta 1 treatment. Recovery of m2 muscarinic receptor mRNA after TGF-beta 1 treatment was slow, with a half-life of approximately 8 hr. There was no effect of TGF-beta 1 on the m2 muscarinic receptor mRNA half-life measured in the presence of actinomycin D, but the rate of m2 muscarinic receptor gene transcription measured with nuclear run-on assay was reduced by 50%, indicating reduced gene transcription. Cycloheximide (10 micrograms/ml) pretreatment abolished the TGF-beta 1 effect, indicating that de novo protein synthesis was required for receptor downregulation. In summary, we have shown that TGF-beta 1 induced desensitization and down-regulation of M2 muscarinic receptor protein and gene that was mediated through reduction in the rate of m2 receptor gene transcription.

Adenylyl Cyclases↗
PubMed1995

Transcriptional down-regulation of m2 muscarinic receptor gene expression in human embryonic lung (HEL 299) cells by protein kinase C.

m2 muscarinic receptor gene expression was investigated following stimulation of protein kinase C (PKC) with the phorbol ester 4 beta-phorbol dibutyrate (PDBu) in HEL 299 cells. PDBu (100 nM) caused a time-dependent decrease in the steady-state levels of m2 receptor mRNA and in specific [3H]N-methyl-scopolamine binding. Preincubation with the PKC inhibitor GF-109203X inhibited the reduction in M2 receptor and mRNA levels induced by PDBu, confirming the involvement of PKC. Chronic PDBu treatment also caused desensitization of the receptor as forskolin-stimulated cAMP accumulation, inhibited by carbachol in control cells, was lost upon treatment with PDBu for 24 h. Co-incubation with PDBu and the protein synthesis inhibitor cycloheximide, inhibited PDBu-mediated reduction of m2 receptor mRNA, indicating new protein synthesis is required for down-regulation. Half-life studies using the transcriptional inhibitor actinomycin D suggested that the stability of the m2 receptor mRNA was not altered by PDBu treatment (t1/2 = 2 h). Nuclear run-on assays showed a 50% reduction in the rate of m2 receptor gene transcription after treatment with PDBu for 12 h. In conclusion we have provided evidence for heterologous regulation of m2 receptor gene expression through changes in gene transcription resulting in uncoupling of M2 receptors. Furthermore, the synthesis of an unidentified factor is required for the down-regulation process.

Blotting, Northern↗
PubMed1995

Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist for the treatment of obstructive airways disease.

Tiotropium bromide (Ba 679 BR) is a novel potent and long-lasting muscarinic antagonist that has been developed for the treatment of chronic obstructive airways disease (COPD). Binding studies with [3H]tiotropium bromide in human lung have confirmed that this is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. Tiotropium bromide dissociates very slowly from lung muscarinic receptors compared with ipratropium bromide. In vitro tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. After washout, however, tiotropium bromide dissociates extremely slowly compared with the dissociation of atropine and ipratropium bromide. Measurement of acetylcholine (ACh) release from guinea-pig trachea shows that tiotropium bromide, ipratropium bromide and atropine all increase ACh release on neural stimulation and that this effect is washed out equally quickly for the three antagonists. This confirms binding studies to transfected human muscarinic receptors which suggested that tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Clinical studies with inhaled tiotropium bromide confirm that it is a potent and long-lasting bronchodilator in COPD and asthma. Furthermore, it protects against cholinergic bronchoconstriction for > 24 h. This suggests that tiotropium bromide will be a useful bronchodilator, particularly in patients with COPD, and may be suitable for daily dosing. The selectivity for M3- over M2-receptors may also confer a clinical advantage.

Acetylcholine↗
PubMed1995

Characterization and autoradiographic mapping of [3H]CP96,345, a nonpeptide selective NK1 receptor antagonist in guinea pig lung.

We have studied binding and distribution of NK1 receptors in guinea pig lung using [3H]CP96,345. Kinetic studies showed that specific binding of [3H]CP96,345 was rapid and reversible, giving a kinetic dissociation constant (Kd) of 0.28 +/- 0.05 nM. The specific binding was also saturable and Scatchard analysis indicated a single class of binding site with an equilibrium Kd of 0.12 +/- 0.03 nM and maximum binding capacity (Bmax) of 107.0 +/- 10.3 fmol/mg of protein. Competition studies showed the rank order of affinity for agonists and antagonists as follows: SP > NKA = septide >> NKB = senktide; CP96,345 > FK888 > FK224 > L668169. NK3 agonists, NK2-selective antagonists, and a calcium channel blocker, diltiazem, showed no displacement, indicating high selectivity for NK1 receptors. Autoradiographic mapping showed specific labeling over airway smooth muscle from central to peripheral airways, submucosal glands, and nerve fibers of trachea. The labeling of airway epithelium was increased with diminishing size of airways. Pulmonary blood vessels were also moderately labeled and there was sparse labeling over alveolar walls. [3H]CP96,345 may provide a useful tool to evaluate NK1 receptor expression in peripheral organs.

Animals↗
PubMed1995

Long-term carbachol treatment-induced down-regulation of muscarinic M2-receptors but not m2 receptor mRNA in a human lung cell line.

1. The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. We have investigated the effect of homologous stimulation on the regulation of M2 muscarinic receptor protein and gene in human embryonic lung fibroblasts (HEL 299 cells). 2. Saturation studies performed with the non-selective hydrophilic ([3H]-N-methyl-scopolamine, [3H]-NMS) and lipophilic (3H]-quinuclidinyl benzilate, [3H]-QNB) muscarinic antagonists revealed a single class of high affinity binding sites. 3. Carbachol (1 mM) induced a rapid down-regulation of [3H]-NMS binding sites. Within 12 h, the process had approached steady state with 40 to 60% loss of receptors at 12 and 24 h. 4. The loss of [3h]-QNB binding sites (40% reduction at 24 h) occurred at a slower rate than did loss of [3H]-NMS binding sites as a result of receptor sequestration. 5. Carbachol treatment was accompanied by a functional desensitization of the receptor after 24 h of agonist treatment. In untreated cells, forskolin induced a large increase in cyclic AMP accumulation which was inhibited significantly by carbachol. The inhibitory effect of carbachol on forskolin-induced cyclic AMP accumulation was lost following 24 h carbachol stimulation. 6. The steady state level of muscarinic m2 mRNA measured by Northern blot analysis was not affected by carbachol had no effect on the stability of m2 mRNA. 7. The rate of transcription of m2 muscarinic receptor gene as measured by nuclear RNA run-on assay was unaltered by carbachol stimulation. 8. These results suggest that homologous sequestration, desensitization, and down-regulation of M2 modifications of m2 muscarinic receptor mRNAs.

Binding, Competitive↗
PubMed1995

Glucocorticosteroids increase beta 2-adrenergic receptor transcription in human lung.

beta 2-Adrenergic receptors (beta 2R) are widely distributed and mediate a wide range of cellular responses in lung. Because glucocorticosteroids increase expression of beta 2R in cell lines, we have investigated the effects of glucocorticoids on the beta 2R mRNA level and the number of beta 2R in human peripheral lung in vitro. Incubation of lung tissues with dexamethasone (Dex) elevated both beta 2R mRNA level (as measured by Northern blot analysis) and beta 2R number (as measured by [125I]iodocyanopindolol binding). The increased accumulation of beta 2R mRNA could be detected at 15 min (1.27 +/- 0.1-fold) and the maximal accumulation occurred at 2 h (2.73 +/- 0.5-fold). The Dex-induced increase in beta 2R mRNA returned to the control level by 17 h. The increase in beta 2R number (1.58 +/- 0.2-fold) was slower, reaching a maximum between 17 and 24 h. Dex increased beta 2R mRNA in a time- and concentration-dependent manner that was abolished by the steroid receptor antagonist mifepristone (RU-38486 or RU-486). The stability of beta 2R mRNA was unchanged by Dex, and a nuclear run-on assay revealed that Dex approximately doubled the transcriptional rate of the beta 2R gene. These observations suggest that glucocorticoids act on steroid receptors to increase beta 2R expression by increasing the rate of beta 2R gene transcription.

Adolescent↗
PubMed1995

Protective effects of a glucocorticoid on downregulation of pulmonary beta 2-adrenergic receptors in vivo.

We investigated the in vivo effects of a glucocorticoid on beta-agonist-induced downregulation of beta 1- and beta 2-adrenergic receptors (determined by [125I]iodocyanopindolol binding), mRNA expression (assessed by Northern blotting), and gene transcription (using nuclear run-on assays) in rat lung. Dexamethasone (Dex) (0.2 mg/kg/d, days 1-8) increased beta 1- and beta 2-receptor numbers by 70 and 69% above control, respectively, but did not change their mRNA expression. Isoproterenol (Iso) (0.96 mg/kg/d, days 2-8) decreased beta 1- and beta 2-receptor numbers by 48 and 51%, respectively, and also reduced mRNA expression by 69 and 57%, respectively. The combination of Dex and Iso resulted in no net change in beta 2-receptor number and its mRNA expression, although there was a significant reduction in beta 1-receptor number and mRNA expression. The mapping of beta 1- and beta 2-receptors by receptor autoradiography confirmed these findings over alveoli, epithelium, endothelium, and airway and vascular smooth muscle. We also measured the activation of the transcription factor, cyclic AMP response element binding protein (CREB) using an electrophoretic mobility shift assay. CREB-like DNA-binding activity was decreased after Iso treatment but this decrease was prevented after treatment with Dex. Nuclear run-on assays revealed that the transcription rate of the beta 1-receptor gene did not alter after Dex treatment, but was reduced after Iso treatment. The transcription rate of the beta 2-receptor gene was increased after Dex treatment by approximately twofold, but there was no change after Iso treatment. We conclude that glucocorticoids can prevent homologous downregulation of beta 2-receptor number and mRNA expression at the transcriptional level without affecting beta 1-receptors and that the transcription factor CREB may be involved in this phenomenon. Such an effect may have clinical implications for preventing the development of tolerance to beta 2-agonists in asthmatic patients treated with beta-agonist bronchodilators.

Animals↗
PubMed1994

High affinity [3H]formoterol binding sites in lung: characterization and autoradiographic mapping.

Agonist binding to the beta 2-adrenoceptors and its mapping were studied using the newly developed radioligand [3H]formoterol. The results of [3H]formoterol saturation binding and formoterol inhibition of [3H]formoterol binding were consistent with binding to a single class of receptors (Kd = 1.34 +/- 0.15 nM, Bmax = 154.9 +/- 8.0 fmol/mg protein in guinea pig lung membranes, n = 8; Kd = 1.05 +/- 0.17 nM, Bmax = 67.8 +/- 8.1 fmol/mg protein in human lung membranes, n = 5) and competition assays with other agonists and antagonists disclosed only a single class of site. The nonhydrolyzable GTP analogue GTP gamma S caused a reduction in both Kd and Bmax, indicating that the receptors labelled by [3H]formoterol are coupled to a guanine nucleotide binding regulatory protein. Receptor mapping of [3H]formoterol binding sites shows that beta 2-adrenoceptors were widely distributed in both guinea pig and human lung, with dense labelling over airway epithelium and uniformly over alveolar walls, and sparse labelling of airway and vascular smooth muscle. In addition, submucosal glands were also sparsely labelled in human bronchus. The distribution of beta 2-adrenoceptors was similar to the pattern previously described with non-selective radiolabelled antagonists in the presence of selective beta 1-adrenoceptor antagonists.

Adrenergic beta-Agonists↗