Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice.

Bakground/Objectives:Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug. SUBJECTS/METHODS: Small molecules modulating SR protein activity and splicing were tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in diet-induced obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet. RESULTS: We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished DIO in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of white adipose tissue from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids. CONCLUSIONS: Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.

Asymptomatic individuals with high HDL-C levels overexpress ABCA1 and ABCG1 and present miR-33a dysregulation in peripheral blood mononuclear cells.

Considering the growing knowledge and perspectives on microRNAs (miRNAs) that control high-density lipoprotein cholesterol (HDL-C) levels and metabolism, this study aimed at evaluating whether hsa-miR-33a and hsa-miR-128a are differentially expressed in peripheral blood mononuclear cells from asymptomatic individuals with low and high HDL-C, as well as at investigating the potential relationships with ATP binding cassete transporter A1 (ABCA1) expression, cholesterol efflux capacity and other parameters related with reverse cholesterol transport. In addition, the associations with cardiovascular risk were investigated by carotid-intima media thickness (cIMT). Asymptomatic volunteers of both genders (n=51) were classified according to HDL-C (mg/dL) in hypoalphalipoproteinemics (hypo, HDL-C ≤3 9), hyperalphalipoproteinemics (hyper, HDL-C ≥ 68) and controls (CTL, HDL-C ≥ 40<68). cIMT, lipids, lipoproteins, HDL size and volume, C reactive protein and insulin were determined, as well as the activities of several proteins and enzymes related to HDL metabolism. In a subgroup of 19 volunteers the cellular cholesterol efflux and HDL composition were determined. Total RNA was extracted from peripheral blood mononuclear cells for relative quantification experiments. Hypo volunteers presented significantly higher levels of triglycerides, VLDL-C and insulin; in addition, HDL size and volume decreased when compared with CTL and hyper. Regarding gene expression analysis, the hyper group presented a decrease of 72% in hsa-miR-33a and higher mRNA expression of ABCA1 and ABCG1 when compared with CTL. No significant differences in hsa-miR-128a expression, cholesterol efflux, cIMT or plaques were found. Further studies are necessary to elucidate the mechanisms underlying the complex miRNA network, regulating cellular cholesterol homeostasis in humans and its clinical repercussions.

Reference values for high-density lipoprotein particle size and volume by dynamic light scattering in a Brazilian population sample and their relationships with metabolic parameters.

BACKGROUND: Current data indicate that the size of high-density lipoprotein (HDL) may be considered an important marker for cardiovascular disease risk. We established reference values of mean HDL size and volume in an asymptomatic representative Brazilian population sample (n=590) and their associations with metabolic parameters by gender. METHODS: Size and volume were determined in HDL isolated from plasma by polyethyleneglycol precipitation of apoB-containing lipoproteins and measured using the dynamic light scattering (DLS) technique. RESULTS: Although the gender and age distributions agreed with other studies, the mean HDL size reference value was slightly lower than in some other populations. Both HDL size and volume were influenced by gender and varied according to age. HDL size was associated with age and HDL-C (total population); non- white ethnicity and CETP inversely (females); HDL-C and PLTP mass (males). On the other hand, HDL volume was determined only by HDL-C (total population and in both genders) and by PLTP mass (males). CONCLUSIONS: The reference values for mean HDL size and volume using the DLS technique were established in an asymptomatic and representative Brazilian population sample, as well as their related metabolic factors. HDL-C was a major determinant of HDL size and volume, which were differently modulated in females and in males.

Arterial tissue and plasma concentration of enzymatic-driven oxysterols are associated with severe peripheral atherosclerotic disease and systemic inflammatory activity.

INTRODUCTION: Cholesterol undergoes oxidation via both enzymatic stress- and free radical-mediated mechanisms, generating a wide range of oxysterols. In contrast to oxidative stress-driven metabolites, enzymatic stress-derived oxysterols are scarcely studied in their association with atherosclerotic disease in humans. METHODS: 24S-hydroxycholesterol (24S-HC), 25-hydroxycholesterol (25-HC), and 27-hydroxycholesterol (27-HC) were assessed in plasma and arteries with atherosclerotic plaques from 10 patients (54-84 years) with severe peripheral artery disease (PAD) as well as arteries free of atherosclerotic plaques from 13 individuals (45-78 years, controls). RESULTS: Plasma 25-HC was higher in PAD individuals than in controls (6.3[2] vs. 3.9[1.9] ng/mgCol; p = 0.004). 24S-HC and 27-HC levels were, respectively, five- and 20-fold higher in the arterial tissue of PAD individuals than in those of the controls (p = 0.016 and p = 0.001). Plasma C-reactive protein correlated with plasma 24-HC (r = 0.51; p = 0.010), 25-HC (r = 0.75; p < 0.001), 27-HC (r = 0.48; p = 0.015), and with tissue 24S-HC (r = 0.4; p = 0.041) and 27-HC (r = 0.46; p = 0.023). CONCLUSION: Arterial intima accumulation of 27-HC and 24S-HC is associated with advanced atherosclerotic disease and systemic inflammatory activity in individuals with severe PAD.

Association between ABCG1 polymorphism rs1893590 and high-density lipoprotein (HDL) in an asymptomatic Brazilian population.

ATP binding cassette transporter G1 (ABCG1) promotes lipidation of nascent high-density lipoprotein (HDL) particles, acting as an intracellular transporter. SNP rs1893590 (c.-204A > C) of ABCG1 gene has been previously studied and reported as functional over plasma HDL-C and lipoprotein lipase activity. This study aimed to investigate the relationships of SNP rs1893590 with plasma lipids and lipoproteins in a large Brazilian population. Were selected 654 asymptomatic and normolipidemic volunteers from both genders. Clinical and anthropometrical data were taken and blood samples were drawn after 12 h fasting. Plasma lipids and lipoproteins, as well as HDL particle size and volume were determined. Genomic DNA was isolated for SNP rs1893590 detection by TaqMan(®) OpenArray(®) Real-Time PCR Plataform (Applied Biosystems). Mann-Whitney U, Chi square and two-way ANOVA were the used statistical tests. No significant differences were found in the comparison analyses between the allele groups for all studied parameters. Conversely, significant interactions were observed between SNP and age over plasma HDL-C, were volunteers under 60 years with AA genotype had increased HDL-C (p = 0.048). Similar results were observed in the group with body mass index (BMI) < 25 kg/m(2), where volunteers with AA genotype had higher HDL-C levels (p = 0.0034), plus an increased HDL particle size (p = 0.01). These findings indicate that SNP rs1893590 of ABCG1 has a significant impact over HDL-C under asymptomatic clinical conditions in an age and BMI dependent way.

Clinical evaluation of a commercial surface-imaging system for patient positioning in radiotherapy.

BACKGROUND: Laser scanning-based patient surface positioning and surveillance may complement image-guided radiotherapy (IGRT) as a nonradiation-based approach. We investigated the performance of an optical system compared to standard kilovoltage cone-beam computed tomography (CBCT) and its potential to reduce the number of daily CBCTs. PATIENTS AND METHODS: We analyzed the patient positioning of 153 treatment fractions in 21 patients applied to three different treatment regions. Patients were first scanned with CBCT, shifted to the optimal isocenter position, and an optical scan was performed to verify the matching in relation to CBCT. RESULTS: For the head-and-neck region, the lateral/longitudinal/vertical/rotational/roll and pitch shift was 0.9 ± 1.8 mm/-2.7 ± 3.8 mm/-0.8 ± 3.6 mm/0.0 ± 1.1°/-0.5 ± 2.1°/0.2 ± 1.6°. For the thorax, the lateral/longitudinal/vertical/roll and pitch shift was -1.2 ± 3.6 mm/0.8 ± 5.1 mm/0.8 ± 4.3 mm/0.6 ± 1.4°/0.1 ± 0.9°/0.3 ± 1.0°. For the pelvis, the respective values were -2.5 ± 4.1 mm/4.6 ± 7.3 mm/-5.1 ± 7.4 mm/0.3 ± 1.1°/-0.5 ± 1.0°/0.3 ± 2.1°. In total, the recorded disagreement was -1.0 ± 3.6 mm/1.0 ± 6.3 mm/-1.8 ± 5.9 mm/0.3 ± 1.2°/-0.3 ± 1.5°/0.2 ± 1.7°. CONCLUSION: This analysis showed good agreement between the optical scanner approach and CBCT. The optical system holds potential to ensure precise patient positioning and reduced CBCT frequency in tumor locations with fixed relation to surface structures.

[Report of the consensus workshop on microsurgical training at the 32nd annual meeting of the german-speaking group for microsurgery of the peripheral nerves and vessels in Basel 2010]. / Bericht zum Workshop und Consensusmeeting zur Mikrochirurgischen Ausbildung an der 32ten Jahrestagung der Deutschsprachigen Arbeitsgemeinschaft für Mikrochirurgie der peripheren Nerven und Gefäße (DAM) 2010 in Basel.

After the foundation of a trinational task force to develop quality criteria for a training and educational system in microsurgery at the annual conference of the German-speaking group for microsurgery of the nerves and vessels (DAM) in Erlangen 2009, at the 2010 conference in Basel, a modular educational system was approved and criteria for a basic course were discussed. Before the next annual conference in 2011 these aspects should be clarified and defined in a spring meet-ing.

Phenotypic and genotypic characteristics of Staphylococcus aureus isolates from raw bulk-tank milk samples of goats and sheep.

Two hundred and ninety-three isolates of Staphylococcus aureus obtained from 127 bulk-tank milk samples of goats and sheep from Switzerland were characterised by pheno- and genotypic traits. Of the 293 S. aureus isolates, 193 (65.9%) were egg yolk-negative and 15 (5.1%) were negative for clumping factor and/or protein A determined by a latex agglutinating test system. For 285 isolates, PCR amplification of the 3' end of the coagulase gene showed a single amplicon. Five differently sized PCR products of 500, 580, 660, 740 and 820 bp were distinguished. In 191 isolates (n = 293) staphylococcal enterotoxin (SE) genes were detected: 123 isolates tested positive for SEC gene, 31 for SEG gene, 28 for SEA gene, 26 for SEJ gene, 24 for SEI gene, 4 for SEB gene and 4 for SED gene. Furthermore, 126 isolates were positive for the gene encoding the toxic shock syndrome toxin 1. Coagulase gene restriction profile analysis of the 145 isolates harbouring SEA or SEC genes revealed six different patterns using AluI and five different patterns using HaeIII. In summary, within these two groups, high genotypic uniformity within the different sized coagulase gene amplicons was demonstrated. This is the first study providing comprehensive characterisation data of S. aureus strains originating from bulk-tank milk samples of goats and sheep. Remarkable differences in phenotypic traits between S. aureus originating from goats and sheep and bovine milk were found. Moreover, the high prevalence of toxin-producing S. aureus may be important as it is relevant to food hygiene.

Prolonged procoagulant activity on overstretch-injured coronary arteries in pigs.

This study was designed to assess the time course and nature of the vascular procoagulant response after 1.5-fold balloon overstretch injury of the coronary arteries in pigs. Arteries were excised for chromogenic assay of bound factor (F)Xa and thrombin at 24 h, 3 days, 1 week, or 2 weeks after injury. FXa at the site of injury remained elevated for 1 week (4.9 +/- 5.9 microg cm(-2), n = 10), compared with non-injured control arteries (0.4 +/- 0.2 microg cm(-2), n = 18, P = 0.00025), while thrombin was increased only at 24 h. Tissue factor protein was abundant in non-injured coronaries (10 +/- 6 ng microg(-1) total protein, n = 9) and levels were unchanged by injury (13 +/- 11 ng microg(-1), n = 6) or 24-h administration of tissue factor pathway inhibitor (16 +/- 6 ng microg(-1), n = 6). Persistent tissue factor-mediated procoagulant activity may explain the need for prolonged anticoagulation to attenuate neointimal formation after balloon-induced coronary injury.

In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles.

Molecular imaging permits tissues to be functionally characterized by identification of specific cell-surface receptors with targeted contrast agents. In our study, a ligand-targeted acoustic nanoparticle system was used to identify the angioplasty-induced expression of tissue factor by smooth muscle cells within the tunica media. Pig carotid arteries were overstretched bilaterally with balloon catheters, treated with a tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound (20 MHz) before and after treatment. Carotid wall acoustic reflectivities were unaffected by overstretch injury. Tissue factor-targeted nanoemulsion bound and increased the echogenicity of smooth muscle cells expressing tissue factor within the tunica media. The targeted emulsion increased the arterial wall gray scale (99.4+/-14.5; P<.05) relative to pretreatment (41.8+/-11.1, P<0.05) and the control gray scale (pre-emulsion: 49.3+/-9.5; post-emulsion: 43.7+/-6.4; P<.05). The area of acoustic enhancement appeared to coincide with expression of induced tissue factor in the tunica media confirmed by immunohistochemistry. We have demonstrated that this novel nanoemulsion can infiltrate into arterial walls after balloon injury and localize the expression of overstretch-induced tissue factor within pig carotid arteries. Molecular imaging and quantification of complex, biochemical change, such as tissue factor expression after angioplasty, may prove to be a prognostically important predictor of subsequent restenosis.

Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound.

RATIONALE AND OBJECTIVES: Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS: Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS: Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS: The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

Tissue factor pathway inhibitor attenuates procoagulant activity and upregulation of tissue factor at the site of balloon-induced arterial injury in pigs.

Intravenous infusion of recombinant tissue factor pathway inhibitor (rTFPI) for 24 hours decreases neointimal thickening and luminal stenosis 1 month after balloon-induced injury to the carotid arteries in minipigs. This study was designed to determine whether the effect of rTFPI is accounted for by early decreases in procoagulant activity and thrombosis on the injured vessel wall. Vascular injury was induced by balloon hyperinflations in both carotid arteries of anesthetized pigs given no anticoagulant as a control (n=16), an intravenous infusion for 24 hours of rTFPI (0.5 mg/kg bolus and 25 microg. kg(-1). min(-1), n=14), or an intravenous infusion of unfractionated heparin (100 U. kg(-1). h(-1), n=19). Accumulation of radiolabeled autologous platelets was markedly decreased over 24 hours on injured arteries from animals given rTFPI (0.6x10(6)/cm(2)) compared with controls (2.5x10(6)/cm(2), P=0.0004). Deposition of radiolabeled fibrin was also decreased in rTFPI-treated animals (269+/-266 microg/cm(2)) compared with controls (2389+/-1673 microg/cm(2), P=0.04). Similar effects were observed with heparin. However, factor Xa activity, assayed after 24 hours by incubation of the injured arterial segments with the chromogenic substrate S-2222, was decreased more markedly on arteries from rTFPI-treated animals (0.14+/-0.13 OD) than those from heparin-treated animals (0.29+/-0.18 OD) compared with controls (0. 47+/-0.24 OD, P=0.0007). In addition, arteries from rTFPI-treated animals showed a 4-fold lower induction of tissue factor protein compared with controls (P=0.0002). Attenuation of procoagulant activity and tissue factor-mediated thrombin generation in response to injury may account for the promising results with rTFPI in the porcine angioplasty model.

Comparison of methods for local delivery of tissue factor pathway inhibitor to balloon-injured arteries in rabbits.

BACKGROUND: Prolonged intravenous infusions of recombinant tissue factor pathway inhibitor (rTFPI) have been shown to attenuate markedly neointimal formation and stenosis after balloon-induced injury to the carotid arteries in minipigs. DESIGN: Because local delivery of rTFPI to the injury site would be clinically advantageous, we designed this study to compare the local delivery and retention of rTFPI in balloon-injured arteries using three catheter-based systems. METHODS: Similar amounts (range 3-4.5 mg) of a mixture of 125I-labeled and unlabeled rTFPI were delivered by either passive diffusion at moderate pressure (5 x 10(5) Pa with the LocalMed InfusaSleeve, or 4 x 10(5) Pa with the SciMed Dispatch device), or facilitated diffusion combining lower pressure (2 x 10(5) Pa) and electrical current (3.5 mA/cm2; e-MED, iontophoresis) to balloon-injured carotid arteries in anesthetized rabbits. RESULTS: Comparable amounts of rTFPI were retained on the injured vessels immediately after delivery (t = 0) with the LocalMed (628 +/- 68 micrograms/g per cm2, n = 4), SciMed (522 +/- 167 micrograms/g per cm2, n = 4), and e-MED (497 +/- 142 micrograms/g per cm2, n = 4) catheters (NS). However, rTFPI was decreased by 37% after 24 h compared with t = 0 (P < 0.02) in the e-MED group, but was increased 1.5-fold (P = 0.02) and 1.3-fold in the SciMed and LocalMed groups, respectively, presumably because of redistribution of rTFPI from remote endothelial or perivascular sites. Retention of rTFPI was six to nine times higher for injured compared with non-injured arteries, and persisted for at least 48 h after delivery with the LocalMed catheter. CONCLUSIONS: Sustained, marked retention of rTFPI delivered locally at the site of balloon-induced arterial injury appears to result from catheter-based systems that use passive diffusion at moderate pressure.

Glucocorticoids increase bradykinin B2 receptor gene transcription in cultured guinea-pig tracheal smooth muscle cells.

We investigated the effect of the glucocorticoid methylprednisolone on the modulation of the expression of the bradykinin B2 receptors in cultured, guinea-pig, tracheal, smooth muscle cells. These receptors are implicated in the pathogenesis of human asthma. Untreated cells expressed a single population of binding sites for [3H]bradykinin with a dissociation constant, Kd, of 87.7+/-12.0 pM and a maximum binding site density, Bmax, of 245.4+/-71 fmol/mg protein. Treatment of the cultured guinea-pig tracheal smooth muscle cells with methylprednisolone 10(-5) M for 6 h increased the number of bradykinin receptors; this response reached a maximum of 78% and returned to the basal value after 12 h. Bradykinin (10(-12) M) elicited a six-fold higher calcium level in treated cells than in control cells. To investigate bradykinin B2 receptor mRNA expression in guinea-pig cells, we used the reverse transcription polymerase chain reaction (RT-PCR) technique to synthesize a specific bradykinin B2 cDNA probe of 296 bp corresponding to nucleotides 456-751 of the human sequence. This guinea-pig cDNA had 88%, 86% and 83% homology with the corresponding human, mouse and rat sequences, respectively, but no homology with any other known sequences. Following methylprednisolone treatment, Northern blot hybridization indicated that mRNA increased fourfold after 3 h compared with control cells, and returned to basal level within 7 h. The rate of gene transcription, assessed by nuclear run-on assays, increased fourfold after 3 h treatment with 10(-5) M methylprednisolone. These results indicate that glucocorticoids induce early up-regulation of bradykinin B2 receptors in cultured guinea-pig tracheal smooth muscle cells by increasing the rate of transcription of the bradykinin B2 receptor gene.

Glucocorticoids inhibit the bradykinin B2 receptor increase induced by interleukin-1beta in human bronchial smooth muscle cells.

We studied the effect of the glucocorticoids, dexamethasone and budesonide, on the interleukin-1beta-induced increase of bradykinin B2 receptors in cultured human bronchial smooth muscle cells, a cellular model of bronchial hyperreactivity. Both compounds prevented the increase of the bradykinin B2 mRNA and the bradykinin-induced inositol phosphate accumulation. These results demonstrate a direct effect of glucocorticoids on airway smooth muscle hyperresponsiveness mediated through inhibition of the over-expression of receptors for contractile mediators induced by inflammatory mediators.

Interleukin-1beta induces bradykinin B2 receptor gene expression through a prostanoid cyclic AMP-dependent pathway in human bronchial smooth muscle cells.

We investigated the hypothesis that inflammatory mediators such as interleukin-1beta (IL-1beta) might be responsible for the hyperreactivity of asthmatic patients to bradykinin. In cultured human bronchial smooth muscle cells, IL-1beta elicited a rapid and transient increase in the density of bradykinin B2 receptors without affecting their affinity for ligands. The increase in B2 receptors was correlated to an enhancement of inositol phosphate formation elicited by bradykinin, indicating its relevance to the contractile response of smooth muscle cells to bradykinin. The increase in receptor density was related to an increase in B2 receptor mRNA level corresponding to a 5-fold enhancement of the transcriptional rate and to a lengthened half-life of mRNA. These effects of IL-1beta were largely inhibited by indomethacin, suggesting the involvement of a prostanoid pathway in IL-1beta transduction process. An increase in prostaglandin E2 levels preceded the mRNA increase, confirming this involvement. Moreover, IL-1beta and prostaglandin E2 led to cAMP formation. We propose this predominant transduction pathway of IL-1beta to stimulate the transcription of the bradykinin B2 gene in human bronchial smooth muscle cells as a major mechanism involved in the hyperresponsiveness of asthmatic patients to bradykinin.

Effect of WIN 64338, a B2 bradykinin receptor antagonist on guinea-pig tracheal smooth muscle cells in culture.

This study investigated the effect of the non-peptidic B2 bradykinin (BK) receptor antagonist WIN 64338 on BK binding and BK-induced inositol phosphate formation on guinea-pig tracheal smooth muscle cells in culture. The presence of specific and saturable binding sites for BK was demonstrated using [3H]BK. Scatchard analysis indicates a single population of binding sites for [3H]BK with a maximal density (Bmax) of 245.4 +/- 71 fmol/mg of protein and an equilibrium dissociation constant (Kd) of 87.7 +/- 0.12 pM. The order of potency of] B2 BK receptor ligands was Hoe 140 = NPC 17731 > BK > WIN 64338 > D- Arg0[Hyp3, D-Phe7]-BK > > des-Arg9-BK, while B1 BK receptor antagonist, des-Arg9-[Leu8]-BK, was without effect on [3H]BK binding. These results demonstrate the presence of B2 Bk receptors on cultured tracheal smooth muscle cells. The cells were stimulated by BK, and inositol phosphate formation was determined by anion exchange chromatography. The stimulating effect of BK on inositol phosphate formation was concentration dependent (1 nM to 10 microM). The B1 BK agonist des-Arg9-BK did not induce inositol phosphate formation. The order of potency of B2 antagonists to inhibit BK-induced inositol phosphate formation was Hoe 140 = NPC 17731 > WIN 64338 > D-Arg0[Hyp3, D-Phe7]-BK. This study demonstrates that WIN 64338 is able to displace [3H]BK binding and to inhibit B2-BK-induced inositol phosphate formation on cultured guinea-pig tracheal smooth muscle cells.

Glucocorticoid modulation of muscarinic and beta-adrenergic receptors in guinea pig lung.

We investigated the effect of the in vivo treatment of guinea pigs with methylprednisolone, 10 mg/kg daily, on lung muscarinic and beta-adrenergic receptors. Receptor densities were assessed by saturation experiments of tritiated N-methylscopolamine and dihydroalprenolol binding to lung membranes. After 3 h of treatment, methylprednisolone induced a decrease of 19.2% (P < 0.05) of muscarinic receptors but was without effect on beta-adrenergic receptor density. After 24 h, an increase of 39.7% (P < 0.01) and 16.9% (P < 0.05) was observed for muscarinic and beta-adrenergic receptors, respectively. For muscarinic receptors, this increase reached 53.4% (P < 0.01) within 48 h and stayed at this level until 96 h. The increase of beta-adrenergic receptors was maximal (24.9%) after 72 h and returned to the control value after 96 h. The dissociation constant (Kd) values of both ligands were not affected by the glucocorticoid treatment. Functional studies showed that the 96 h treatment did not affect the contractile response of guinea pig lung parenchymal strips to carbachol since the 50% concentration value (EC50) and the maximal contraction value (Emax) were not significatively different from control values. These data show that glucocorticoids control the expression of both muscarinic and beta-adrenergic receptors in guinea pig lung but with different time courses and to a larger extent for muscarinic receptors. The glucocorticoid treatment did not modify the contractile response of lung strips to carbachol, confirming the absence of effect on the affinity of muscarinic receptors and suggesting that the receptor reserve exceed the increase of their density by the steroid.