Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 46
Filter
1.
Biomed J ; 45(5): 776-787, 2022 Oct.
Article in English | MEDLINE | ID: mdl-34666219

ABSTRACT

INTRODUCTION: Organs procured following brain stem death (BSD) are the main source of organ grafts for transplantation. However, BSD is associated with inflammatory responses that may damage the organ and affect both the quantity and quality of organs available for transplant. Therefore, we aimed to investigate plasma and bronchoalveolar lavage (BAL) pro-inflammatory cytokine profiles and cardiovascular physiology in a clinically relevant 6-h ovine model of BSD. METHODS: Twelve healthy female sheep (37-42 Kg) were anaesthetized and mechanically ventilated prior to undergoing BSD induction and then monitored for 6 h. Plasma and BAL endothelin-1 and cytokines (IL-1ß, 6, 8 and tumour necrosis factor alpha (TNF-α)) were assessed by ELISA. Differential white blood cell counts were performed. Cardiac function during BSD was also examined using echocardiography, and cardiac biomarkers (A-type natriuretic peptide and troponin I were measured in plasma. RESULTS: Plasma concentrations big ET-1, IL-6, IL-8, TNF-α and BAL IL-8 were significantly (p < 0.01) increased over baseline at 6 h post-BSD. Increased numbers of neutrophils were observed in the whole blood (3.1 × 109 cells/L [95% confidence interval (CI) 2.06-4.14] vs. 6 × 109 cells/L [95%CI 3.92-7.97]; p < 0.01) and BAL (4.5 × 109 cells/L [95%CI 0.41-9.41] vs. 26 [95%CI 12.29-39.80]; p = 0.03) after 6 h of BSD induction vs baseline. A significant increase in ANP production (20.28 pM [95%CI 16.18-24.37] vs. 78.68 pM [95%CI 53.16-104.21]; p < 0.0001) and cTnI release (0.039 ng/mL vs. 4.26 [95%CI 2.69-5.83] ng/mL; p < 0.0001), associated with a significant reduction in heart contractile function, were observed between baseline and 6 h. CONCLUSIONS: BSD induced systemic pro-inflammatory responses, characterized by increased neutrophil infiltration and cytokine production in the circulation and BAL fluid, and associated with reduced heart contractile function in ovine model of BSD.


Subject(s)
Heart Diseases , Tumor Necrosis Factor-alpha , Sheep , Animals , Female , Tumor Necrosis Factor-alpha/metabolism , Interleukin-8 , Cytokines/metabolism , Brain Stem
2.
J Biomed Sci ; 27(1): 96, 2020 Oct 02.
Article in English | MEDLINE | ID: mdl-33008372

ABSTRACT

BACKGROUND: A lung transplant is the last resort treatment for many patients with advanced lung disease. The majority of donated lungs come from donors following brain death (BD). The endothelin axis is upregulated in the blood and lung of the donor after BD resulting in systemic inflammation, lung damage and poor lung graft outcomes in the recipient. Tezosentan (endothelin receptor blocker) improves the pulmonary haemodynamic profile; however, it induces adverse effects on other organs at high doses. Application of ex vivo lung perfusion (EVLP) allows the development of organ-specific hormone resuscitation, to maximise and optimise the donor pool. Therefore, we investigate whether the combination of EVLP and tezosentan administration could improve the quality of donor lungs in a clinically relevant 6-h ovine model of brain stem death (BSD). METHODS: After 6 h of BSD, lungs obtained from 12 sheep were divided into two groups, control and tezosentan-treated group, and cannulated for EVLP. The lungs were monitored for 6 h and lung perfusate and tissue samples were processed and analysed. Blood gas variables were measured in perfusate samples as well as total proteins and pro-inflammatory biomarkers, IL-6 and IL-8. Lung tissues were collected at the end of EVLP experiments for histology analysis and wet-dry weight ratio (a measure of oedema). RESULTS: Our results showed a significant improvement in gas exchange [elevated partial pressure of oxygen (P = 0.02) and reduced partial pressure of carbon dioxide (P = 0.03)] in tezosentan-treated lungs compared to controls. However, the lungs hematoxylin-eosin staining histology results showed minimum lung injuries and there was no difference between both control and tezosentan-treated lungs. Similarly, IL-6 and IL-8 levels in lung perfusate showed no difference between control and tezosentan-treated lungs throughout the EVLP. Histological and tissue analysis showed a non-significant reduction in wet/dry weight ratio in tezosentan-treated lung tissues (P = 0.09) when compared to control. CONCLUSIONS: These data indicate that administration of tezosentan could improve pulmonary gas exchange during EVLP.


Subject(s)
Endothelin Receptor Antagonists/pharmacology , Lung/drug effects , Pyridines/pharmacology , Respiratory Function Tests , Tetrazoles/pharmacology , Vasodilator Agents/pharmacology , Animals , Disease Models, Animal , Lung/physiology , Perfusion , Sheep, Domestic , Tissue Donors
3.
Osteoporos Int ; 31(1): 31-42, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31471664

ABSTRACT

Individuals with low socio-economic status (SES) have a higher risk of dying following hip fracture compared with individuals with high SES. Evidence on social inequalities in non-hip fractures is lacking as well as evidence on the impact of SES on health-related quality of life post fracture. INTRODUCTION: Fragility fractures, especially of the hip, cause substantial excess mortality and impairment in health-related quality of life (HRQoL). This systematic review and meta-analysis aimed to investigate the association between socio-economic status (SES) and post-fracture mortality and HRQoL. METHODS: PubMed, EMBASE and CINAHL databases were searched from inception to the last week of November 2018 for studies reporting an association between SES and post-fracture mortality and/or HRQoL among people aged ≥ 50 years. Risk ratios (RRs) were meta-analyzed using a standard inverse-variance-weighted random effects model. Studies using individual-level and area-based SES measures were analyzed separately. RESULTS: A total of 24 studies from 15 different countries and involving more than one million patients with hip fractures were included. The overall risk of mortality within 1-year post-hip fracture in individuals with low SES was 24% higher than in individuals with high SES (RR 1.24, 95% CI 1.19 to 1.29) for individual-level SES measures, and 14% (RR 1.14, 95% CI 1.09 to 1.19) for area-based SES measures. The quality of the evidence for the outcome mortality was moderate. Using individual SES measures, we estimated the excess HRQoL loss to be 5% (95% CI - 1 to 10%) among hip fracture patients with low SES compared with high SES. CONCLUSIONS: We found a consistently increased risk of post-hip fracture mortality with low SES across SES measures and across countries with different political structures and different health and social care infrastructures. The impact of SES on post-fracture HRQoL remains uncertain due to sparse and low-quality evidence.


Subject(s)
Frailty , Health Status Disparities , Hip Fractures , Quality of Life , Aged , Female , Humans , Prognosis , Socioeconomic Factors
4.
Eur Respir J ; 31(1): 143-78, 2008 01.
Article in English | MEDLINE | ID: mdl-18166595

ABSTRACT

Asthma is a serious health problem throughout the world. During the past two decades, many scientific advances have improved our understanding of asthma and ability to manage and control it effectively. However, recommendations for asthma care need to be adapted to local conditions, resources and services. Since it was formed in 1993, the Global Initiative for Asthma, a network of individuals, organisations and public health officials, has played a leading role in disseminating information about the care of patients with asthma based on a process of continuous review of published scientific investigations. A comprehensive workshop report entitled "A Global Strategy for Asthma Management and Prevention", first published in 1995, has been widely adopted, translated and reproduced, and forms the basis for many national guidelines. The 2006 report contains important new themes. First, it asserts that "it is reasonable to expect that in most patients with asthma, control of the disease can and should be achieved and maintained," and recommends a change in approach to asthma management, with asthma control, rather than asthma severity, being the focus of treatment decisions. The importance of the patient-care giver partnership and guided self-management, along with setting goals for treatment, are also emphasised.


Subject(s)
Asthma/diagnosis , Asthma/prevention & control , Asthma/therapy , Adrenal Cortex Hormones/pharmacology , Anti-Asthmatic Agents/therapeutic use , Asthma/epidemiology , Diagnosis, Differential , Disease Management , Global Health , Guidelines as Topic , Humans , Interdisciplinary Communication , Public Health , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Medicine/methods , Risk Factors
5.
Eur Respir J ; 29(1): 56-62, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17050557

ABSTRACT

The present study examined the association between guideline-derived asthma control and health-related quality of life, assessed using the Asthma Quality of Life Questionnaire (AQLQ), in patients with uncontrolled asthma whose treatment was directed towards achieving the highest possible level of control. The present randomised, double-blind, parallel-group study compared the efficacy of fluticasone propionate (FP) and salmeterol/fluticasone propionate combination (SFC) in achieving two composite, guideline-derived measures of control: total control (TC) and well-controlled (WC) asthma. Not achieving these levels was classed as not well-controlled (NWC). Doses were augmented until patients achieved TC or reached the maximum dose. This dose was maintained for the remainder of the study. AQLQ was assessed at baseline and at each clinic visit. AQLQ scores improved throughout the study, reaching near-maximal levels in patients achieving TC and WC, and 52-week mean scores in the three control groups were statistically significantly different. Clinically meaningful improvements (mean change from baseline) were: TC group (SFC 1.9, FP 1.8), WC (SFC 1.5, FP 1.5) and NWC (SFC 1.0, FP 0.9). In conclusion, the treatment aimed at controlling asthma improves the health-related quality of life to levels approaching normal. The difference in Asthma Quality of Life Questionnaire scores between total control and well-controlled confirms that patients distinguish even between these high levels of control.


Subject(s)
Albuterol/analogs & derivatives , Androstadienes/administration & dosage , Asthma/drug therapy , Health Status , Quality of Life , Administration, Inhalation , Adolescent , Adult , Albuterol/administration & dosage , Bronchodilator Agents , Double-Blind Method , Drug Combinations , Female , Fluticasone , Fluticasone-Salmeterol Drug Combination , Humans , Male , Metered Dose Inhalers , Middle Aged , Treatment Outcome
6.
Biochim Biophys Acta ; 1758(11): 1723-31, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17069752

ABSTRACT

We describe the phenomenon of mechanoelectrical transduction in macroscopic lipid bilayer membranes modified by two cation-selective ionophores, valinomycin and nonactin. We found that bulging these membranes, while maintaining the membrane tension constant, produced a marked supralinear increase in specific carrier-mediated conductance. Analyses of the mechanisms involved in mechanoelectrical transduction induced by the imposition of a hydrostatic pressure gradient or by an amphipathic compound chlorpromazine reveal similar changes in the charge carrier motility and carrier reaction rates at the interface(s). Furthermore, the relative change in membrane conductance was independent of membrane diameter, but was directly proportional to the square of membrane curvature, thus relating the observed phenomena to the bilayer bending energy. Extrapolated to biological membranes, these findings indicate that ion transport in cells can be influenced simply by changing shape of the membrane, without a change in membrane tension.


Subject(s)
Anti-Bacterial Agents/pharmacology , Cell Membrane Permeability/drug effects , Electric Conductivity , Lipid Bilayers/metabolism , Valinomycin/pharmacology , Biological Transport, Active , Cations, Monovalent , Chlorpromazine/pharmacology , Hydrostatic Pressure , Iontophoresis/methods , Kinetics , Macrolides/pharmacology , Mathematics , Membrane Potentials , Models, Biological , Thermodynamics
7.
Allergy ; 61(5): 531-6, 2006 May.
Article in English | MEDLINE | ID: mdl-16629780

ABSTRACT

BACKGROUND: The Gaining Optimal Asthma ControL (GOAL) study has shown the superiority of a combination of salmeterol/fluticasone propionate (SFC) compared with fluticasone propionate alone (FP) in terms of improving guideline defined asthma control. METHODS: Clinical and economic data were taken from the GOAL study, supplemented with data on health related quality of life, in order to estimate the cost per quality adjusted life year (QALY) results for each of three strata (previously corticosteroid-free, low- and moderate-dose corticosteroid users). A series of statistical models of trial outcomes was used to construct cost effectiveness estimates across the strata of the multinational GOAL study including adjustment to the UK experience. Uncertainty was handled using the non-parametric bootstrap. Cost-effectiveness was compared with other treatments for chronic conditions. RESULT: Salmeterol/fluticasone propionate improved the proportion of patients achieving totally and well-controlled weeks resulting in a similar QALY gain across the three strata of GOAL. Additional costs of treatment were greatest in stratum 1 and least in stratum 3, with some of the costs offset by reduced health care resource use. Cost-effectiveness by stratum was 7600 pound (95% CI: 4800-10,700 pound) per QALY gained for stratum 3; 11,000 pound (8600-14,600 pound) per QALY gained for stratum 2; and 13,700 pound (11,000-18,300 pound) per QALY gained for stratum 1. CONCLUSION: The GOAL study previously demonstrated the improvement in total control associated with the use of SFC compared with FP alone. This study suggests that this improvement in control is associated with cost-per-QALY figures that compare favourably with other uses of scarce health care resources.


Subject(s)
Asthma/drug therapy , Asthma/economics , Bronchodilator Agents/economics , Outcome Assessment, Health Care/economics , Quality-Adjusted Life Years , Albuterol/analogs & derivatives , Albuterol/economics , Albuterol/therapeutic use , Androstadienes/economics , Androstadienes/therapeutic use , Bronchodilator Agents/therapeutic use , Cost-Benefit Analysis/methods , Cost-Benefit Analysis/statistics & numerical data , Double-Blind Method , Drug Combinations , Fluticasone , Fluticasone-Salmeterol Drug Combination , Humans , Models, Statistical , Outcome Assessment, Health Care/methods , Quality of Life , Time Factors , Treatment Outcome , United Kingdom
8.
J Biol Chem ; 276(26): 23589-98, 2001 Jun 29.
Article in English | MEDLINE | ID: mdl-11323431

ABSTRACT

We have tested the importance of charge interactions for alpha-conotoxin MI binding to the nicotinic acetylcholine receptor (AChR). Ionic residues on alpha-conotoxin MI were altered by site-directed mutagenesis or by chemical modification. In physiological buffer, removal of charges at the N terminus, His-5, and Lys-10 had small (2-4-fold) effects on binding affinity to the mouse muscle AChR and the Torpedo AChR. It was also demonstrated that conotoxin had no effect on the conformational equilibrium of either receptor, as assessed by the effects of the noncompetitive antagonist proadifen on conotoxin binding and, conversely, the effect of conotoxin on the affinity of phencyclidine, proadifen, and ethidium. Conotoxin displayed higher binding affinity in low ionic strength buffer; neutralization of Lys-10 and the N terminus by acetylation blocked this affinity shift at the alphadelta site but not at the alphagamma site. It is concluded that Ctx residues Lys-10 and the N terminal interact with oppositely charged receptor residues only at the alphadelta site, and the two sites have distinct arrangements of charged residues. Ethidium fluorescence experiments demonstrated that conotoxin is formally competitive with a small cholinergic ligand, tetramethylammonium. Thus, alpha-conotoxin MI appears to interact with the portion of the binding site responsible for stabilizing agonist cations but does not do so with a cationic residue and is, consequently, incapable of inducing a conformational change.


Subject(s)
Conotoxins/metabolism , Receptors, Nicotinic/metabolism , Animals , Binding Sites , Binding, Competitive , Conotoxins/genetics , Escherichia coli/genetics , Lysine/chemistry , Lysine/genetics , Mice , Mutagenesis, Site-Directed , Osmolar Concentration , Protein Binding , Protein Conformation , Quaternary Ammonium Compounds/metabolism , Receptors, Nicotinic/chemistry , Recombinant Proteins/metabolism , Static Electricity , Torpedo
9.
J Biol Chem ; 276(3): 2069-74, 2001 Jan 19.
Article in English | MEDLINE | ID: mdl-11035044

ABSTRACT

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.


Subject(s)
Calcium/metabolism , Calmodulin/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Amino Acid Sequence , Binding Sites , Fluorescence , Molecular Sequence Data , Protein Binding , Ryanodine Receptor Calcium Release Channel/chemistry , Tryptophan/chemistry
10.
Ugeskr Laeger ; 163(46): 6391-5, 2001 Nov 12.
Article in Danish | MEDLINE | ID: mdl-11816914

ABSTRACT

Paediatric pulmonology (paediatric respiratory medicine) concerns such lung diseases in children as asthma, pneumonia, cystic fibrosis, primary ciliary dyskinesia, chronic interstitial pneumonia, bronchopulmonary dysplasia and congenital abnormalities. This specialty has been approved as an official subsection of the European Confederation of Specialists in Paediatrics (CESP) and acknowledged by the European Union of Medical Specialists (UEMS). A training syllabus has been defined and training centres in all EU countries, including Denmark, have been identified and approved by the local paediatric organisations. The training syllabus emphasises routine in the clinical diagnosis and treatment of the diseases, as well as methods such as lung function in all age groups, bronchoscopy, biopsy, and others. This article summarises the status of this specialty, and the training syllabus, and highlights key research questions.


Subject(s)
Lung Diseases , Pediatrics , Pulmonary Medicine , Asthma/diagnosis , Asthma/therapy , Bronchopulmonary Dysplasia/diagnosis , Bronchopulmonary Dysplasia/therapy , Child , Ciliary Motility Disorders/diagnosis , Ciliary Motility Disorders/therapy , Clinical Competence , Curriculum , Cystic Fibrosis/diagnosis , Cystic Fibrosis/therapy , Europe , Humans , Infant, Newborn , Lung Diseases/congenital , Lung Diseases/diagnosis , Lung Diseases/therapy , Lung Diseases, Interstitial/diagnosis , Lung Diseases, Interstitial/therapy , Pediatrics/education , Pediatrics/organization & administration , Pediatrics/standards , Pneumonia/diagnosis , Pneumonia/microbiology , Pneumonia/therapy , Pulmonary Medicine/education , Pulmonary Medicine/organization & administration , Pulmonary Medicine/standards
11.
Ugeskr Laeger ; 163(48): 6746-50, 2001 Nov 26.
Article in Danish | MEDLINE | ID: mdl-11768900

ABSTRACT

INTRODUCTION: Short-term studies have shown that inhaled corticosteroids may retard the growth of children with asthma. The effect of long-term treatment on adult height is, however, uncertain. MATERIAL AND METHODS: We conducted a prospective study of children with asthma to examine the effect of long-term treatment with inhaled budesonide on adult height. We report on 211 children who have attained adult height: 142 asthmatic children treated with budesonide, 18 asthmatic control patients who have never received inhaled corticosteroids, and 51 healthy siblings of patients in the budesonide group, who also served as controls. RESULTS: The children in the budesonide group attained adult height after a mean of 9.2 years of budesonide treatment (range 3-13 years) at a mean daily dose of 412 micrograms (range 110-877 micrograms). The mean cumulative dose of budesonide was 1.35 g (range 0.41-3.99 g). The mean differences between the measured and target adult heights were +0.3 cm (95% confidence intervals: -0.6; +1.2 cm) for the children treated with budesonide, -0.2 cm (95% confidence intervals: -2.4; +2.1 cm) for the control children with asthma, and +0.9 cm (95% confidence intervals: -0.4; +2.2 cm) for the healthy siblings. The adult height depended significantly (p < 0.001) on the child's height before budesonide treatment. Although growth rates were significantly reduced during the first years of budesonide treatment, these changes were not significantly associated with adult height. DISCUSSIONS: Children with asthma who have received long-term treatment with inhaled budesonide attain normal adult height.


Subject(s)
Asthma/drug therapy , Body Height/drug effects , Bronchodilator Agents/administration & dosage , Budesonide/administration & dosage , Growth/drug effects , Adolescent , Adult , Bronchodilator Agents/adverse effects , Budesonide/adverse effects , Child , Child, Preschool , Female , Humans , Infant , Male , Prospective Studies , Time Factors
12.
Biochemistry ; 39(37): 11452-62, 2000 Sep 19.
Article in English | MEDLINE | ID: mdl-10985791

ABSTRACT

The binding sites of ethidium, a noncompetitive antagonist of the nicotinic acetylcholine receptor (nAChR), have been localized in the Torpedo nAChR in the desensitized state by use of a photoactivatible derivative, [(3)H]ethidium diazide. At 10 microM [(3)H]ethidium diazide, incorporation into the alpha-, beta-, and delta-subunits was inhibited by the presence of phencyclidine (PCP). Within the alpha-subunit, the incorporation was mapped to a 20-kDa fragment beginning at alphaSer-173 and containing the first three transmembrane segments, alphaM1, alphaM2, and alphaM3. Further digestion of this fragment generated two fragments with PCP-inhibitable incorporation, one containing alphaM1 and one containing both alphaM2 and alphaM3. Within alphaM2, specific incorporation was present in alphaLeu-251 and alphaSer-252, residues that have been previously shown to line the lumen of the ion channel. Digestion of the delta-subunit with S. aureus V8 protease generated a 14-kDa and a 20-kDa fragment, both of which began at Ile-192 and contained PCP-inhibitable labeling. The 14-kDa fragment, containing deltaM1 and deltaM2, was further digested to generate a 3-kDa fragment, containing deltaM2 alone, with PCP-inhibitable incorporation. Digestion of the 20-kDa fragment, which contained deltaM1, deltaM2, and deltaM3, generated two fragments with incorporation, one containing the deltaM1 segment and the other containing deltaM2 and deltaM3. These results establish that in the desensitized state of the nAChR, the high-affinity binding site of ethidium is within the lumen of the ion channel and that the bound drug is in contact with amino acids from both the M1 and M2 hydrophobic segments.


Subject(s)
Azides/metabolism , Ethidium/metabolism , Ion Channels/metabolism , Photoaffinity Labels/metabolism , Receptors, Nicotinic/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Membrane/metabolism , Electric Organ/metabolism , Ethidium/analogs & derivatives , Molecular Sequence Data , Peptide Fragments/metabolism , Peptide Mapping , Receptor, Muscarinic M1 , Receptor, Muscarinic M2 , Receptors, Muscarinic/metabolism , Serine Endopeptidases/metabolism , Staphylococcus aureus , Torpedo , Tritium
13.
Biophys J ; 78(3): 1324-34, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10692319

ABSTRACT

To determine the importance of electrostatic interactions for agonist binding to the nicotinic acetylcholine receptor (AChR), we examined the affinity of the fluorescent agonist dansyl-C6-choline for the AChR. Increasing ionic strength decreased the binding affinity in a noncompetitive manner and increased the Hill coefficient of binding. Small cations did not compete directly for dansyl-C6-choline binding. The sensitivity to ionic strength was reduced in the presence of proadifen, a noncompetitive antagonist that desensitizes the receptor. Moreover, at low ionic strength, the dansyl-C6-choline affinities were similar in the absence or presence of proadifen, a result consistent with the receptor being desensitized at low ionic strength. Similar ionic strength effects were observed for the binding of the noncompetitive antagonist [(3)H]ethidium when examined in the presence and absence of agonist to desensitize the AChR. Therefore, ionic strength modulates binding affinity through at least two mechanisms: by influencing the conformation of the AChR and by electrostatic effects at the binding sites. The results show that charge-charge interactions regulate the desensitization of the receptor. Analysis of dansyl-C6-choline binding to the desensitized conformation using the Debye-Hückel equation was consistent with the presence of five to nine negative charges within 20 A of the acetylcholine binding sites.


Subject(s)
Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Animals , Binding Sites , Carbachol/pharmacology , Cell Membrane/metabolism , Dansyl Compounds/pharmacokinetics , Electric Organ/physiology , Ethidium/pharmacokinetics , Fluorescent Dyes , Kinetics , Models, Chemical , Models, Molecular , Nicotinic Agonists/pharmacokinetics , Nicotinic Antagonists/pharmacokinetics , Osmolar Concentration , Protein Conformation , Quaternary Ammonium Compounds/pharmacokinetics , Radioligand Assay , Static Electricity , Torpedo
15.
Mol Pharmacol ; 55(1): 159-67, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9882710

ABSTRACT

A series of aminotriarylmethane dyes were examined for binding to the nicotinic acetylcholine receptor (AChR) from Torpedo californica. Several compounds were found to bind to the noncompetitive antagonist site of the AChR as demonstrated by inhibition of [3H]phencyclidine binding; apparent KD values ranged from 50 nM to >100 microM. One dye with high affinity, crystal violet, revealed a greater than 200-fold fluorescence enhancement upon binding the AChR. Using fluorescence to measure binding, we determined that one crystal violet bound per receptor with a dissociation constant of 100 nM; in the presence of the agonist carbamylcholine this value decreased to 10 nM. The KD for [3H]acetylcholine binding likewise was decreased in the presence of crystal violet. These results are consistent with preferential binding of crystal violet to the desensitized conformation of the AChR. Crystal violet binding blocked agonist-induced 22Na ion efflux from AChR-rich vesicles. It is concluded that crystal violet and other dyes of similar structure bind to the high-affinity noncompetitive antagonist site of the AChR associated with the channel lumen. Because of their optical properties, crystal violet and several of the other homologous dyes are likely to be useful ligands for further characterization of the AChR channel. Structure-activity comparison of the various dyes suggests the importance of nonquaternary nitrogens in binding the pore. Additional steric bulk on amines or at meta positions increase or have neutral effect on affinity, suggesting that steric considerations alone do not limit high affinity for the binding site.


Subject(s)
Coloring Agents/metabolism , Nicotinic Antagonists/metabolism , Receptors, Nicotinic/metabolism , Animals , Binding Sites , Binding, Competitive , Gentian Violet/metabolism , Phencyclidine/metabolism , Protein Conformation , Receptors, Nicotinic/chemistry , Sodium/metabolism , Spectrometry, Fluorescence , Structure-Activity Relationship , Torpedo
16.
Neuropharmacology ; 37(2): 251-7, 1998.
Article in English | MEDLINE | ID: mdl-9680250

ABSTRACT

D-Tubocurarine is a potent competitive antagonist of two members of the ligand-gated ion channel family, the muscle-type nicotinic acetylcholine receptor (AChR) and serotonin type-3 receptor (5HT3R). We have used a series of analogs of D-tubocurarine to determine the effects of methylation, stereoisomerization and halogenation on the interaction of D-tubocurarine with the 5HT3R. The affinities of the analogs for the 5HT3R span a 200-fold concentration range and fall into three broad groups. The first group, with affinity constants (Ki) < 150 nM, consists of D-tubocurarine and analogs modified at the nitrogens or 7' hydroxyl. The fact that these compounds all have high affinity for the 5HT3R suggests that these portions of the ligand do not make interactions with the receptor that are critical for high-affinity binding. The second group, with Ki's in the 1-5 microM range, consists of analogs modified at the 12'-hydroxyl or the adjacent 13'-carbon, which suggests that this portion of the ligand makes interactions that are important for high-affinity binding. The third, very low affinity, group is a compound with altered stereoconfiguration at the 1 carbon, demonstrating the importance of proper configuration of the antagonist in ligand-receptor interactions. For the most part, this pattern of selectivity is similar to that for the AChR, suggesting that the structures of the ligand-binding sites of these two receptors share common structural features.


Subject(s)
Receptors, Serotonin/metabolism , Tubocurarine/analogs & derivatives , Animals , Binding, Competitive , Cell Line , Receptors, Serotonin, 5-HT3 , Structure-Activity Relationship , Tubocurarine/metabolism
17.
Ugeskr Laeger ; 160(15): 2249-52, 1998 Apr 06.
Article in Danish | MEDLINE | ID: mdl-9599520

ABSTRACT

Children's compliance with orally administered medicine is described. Five hundred questionnaires were given to the parents of children at their first visit at the out-patient clinic with a response from 484. Three hundred and ninety-seven children had been treated with oral medication in the form of liquid formulation or tablets, 257 within the previous year. Of these, 43.2% reported difficulties in taking the medication. Only 8.5% of the treatments had been interrupted. The main cause of the problems were the medications' taste and difficulties with swallowing tablets. The problems were more pronounced among the younger children. The administration of tablets was more difficult than the administration of liquids. There was no relationship between the problems and frequency of dosing, duration of treatment, severity of illness, social status of the parents, or the parents' age. In conclusion, prescription of better tasting mixtures and chewable tablets may reduce the problems associated with administering oral medication to children.


Subject(s)
Administration, Oral , Adolescent , Child , Child, Preschool , Humans , Infant , Patient Compliance , Retrospective Studies , Surveys and Questionnaires , Tablets
18.
J Biol Chem ; 272(40): 24891-8, 1997 Oct 03.
Article in English | MEDLINE | ID: mdl-9312090

ABSTRACT

The binding of d-tubocurarine and several of its analogs to the mouse nicotinic acetylcholine receptor (AChR) was measured by competition against the initial rate 125I-alpha-bungarotoxin binding to BC3H-1 cells. The changes in affinity due to methylation or halogenation at various functional groups on d-tubocurarine was measured to both the high affinity (alphagamma-site) and the low affinity site (alphadelta-site). We show that quaternization by methylation of the 2'-N ammonium group enhances the affinity for both the acetylcholine binding sites of mouse AChR, whereas this change does not affect affinity for the Torpedo AChR sites. The effect of N-methylation suggests the presence of interactions with the ammonium moiety that cannot be readily attributed to the known conserved residues thought to stabilize this functional group. Methylation of both the 7'- and 12'-phenols produced net affinity changes at both sites. The changes resulted from contributions at both the 7'- and the 12'-positions; however, these effects were dependent on whether the ammoniums were also methylated. Substitution of bromine or iodine at the 13'-position decreased the affinity considerably to the high affinity alphagamma-site of mouse AChR, whereas the affinity for the Torpedo alphagamma-site was slightly increased. Furthermore, binding to the mouse AChR was unaffected by the conformational state, whereas these ligands strongly preferred the desensitized conformation of the Torpedo AChR. Comparison of binding changes upon 13'-halogenation to the changes in amino acid residues at the ACh binding sites of the mouse and Torpedo AChR shows mouse residue Ile-gamma116 as likely to be involved in interacting with the 13'-position of d-tubocurarine. It is predicted that this residue is involved in the conformational equilibrium between the resting and desensitized conformations.


Subject(s)
Protein Conformation , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Tubocurarine/analogs & derivatives , Tubocurarine/metabolism , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Cell Line , Cell Membrane/metabolism , Electric Organ/metabolism , Kinetics , Methylation , Mice , Molecular Structure , Quaternary Ammonium Compounds , Structure-Activity Relationship , Torpedo , Tubocurarine/chemistry
19.
Biochemistry ; 36(8): 2068-75, 1997 Feb 25.
Article in English | MEDLINE | ID: mdl-9047305

ABSTRACT

Quinacrine is a noncompetitive antagonist of the nicotinic acetylcholine receptor (AChR) which displays severalfold fluorescent enhancement upon binding to AChR-rich membranes from Torpedo californica electric organ. It is demonstrated that the fluorescence enhancement comprises two components: specific interaction at a high-affinity binding site on the AChR, and interaction with the lipid bilayer. The interaction with the lipid bilayer can be attenuated by other noncompetitive antagonists, but at concentrations substantially higher than those required for binding to the AChR. It is further shown that quinacrine can inhibit the binding of [3H]phencyclidine and [3H]ethidium in a manner fully consistent with simple competitive inhibition. The data support a model for high-affinity quinacrine binding to the same, single locus of the acetylcholine receptor as phencyclidine and ethidium. This site is likely within the lumen of the ion channel.


Subject(s)
Ethidium/metabolism , Nicotinic Antagonists/metabolism , Quinacrine/metabolism , Receptors, Nicotinic/metabolism , Torpedo/metabolism , Animals , Binding Sites , Ethidium/pharmacology , Fluorescent Dyes/metabolism , Fluorescent Dyes/pharmacology , Nicotinic Antagonists/pharmacology , Quinacrine/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...