Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 24
Filter
1.
Patient Saf Surg ; 15(1): 24, 2021 Jul 12.
Article in English | MEDLINE | ID: mdl-34253246

ABSTRACT

BACKGROUND: A retained surgical item (RSI) is defined as a never-event and can have drastic consequences on patient, provider, and hospital. However, despite increased efforts, RSI events remain the number one sentinel event each year. Hard foreign bodies (e.g. surgical sharps) have experienced a relative increase in total RSI events over the past decade. Despite this, there is a lack of literature directed towards this category of RSI event. Here we provide a systematic review that focuses on hard RSIs and their unique challenges, impact, and strategies for prevention and management. METHODS: Multiple systematic reviews on hard RSI events were performed and reported using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) and AMSTAR (Assessing the methodological quality of systematic reviews) guidelines. Database searches were limited to the last 10 years and included surgical "sharps," a term encompassing needles, blades, instruments, wires, and fragments. Separate systematic review was performed for each subset of "sharps". Reviewers applied reciprocal synthesis and refutational synthesis to summarize the evidence and create a qualitative overview. RESULTS: Increased vigilance and improved counting are not enough to eliminate hard RSI events. The accurate reporting of all RSI events and near miss events is a critical step in determining ways to prevent RSI events. The implementation of new technologies, such as barcode or RFID labelling, has been shown to improve patient safety, patient outcomes, and to reduce costs associated with retained soft items, while magnetic retrieval devices, sharp detectors and computer-assisted detection systems appear to be promising tools for increasing the success of metallic RSI recovery. CONCLUSION: The entire healthcare system is negatively impacted by a RSI event. A proactive multimodal approach that focuses on improving team communication and institutional support system, standardizing reports and implementing new technologies is the most effective way to improve the management and prevention of RSI events.

2.
Hosp Pediatr ; 11(8): 806-807, 2021 08.
Article in English | MEDLINE | ID: mdl-34244335

ABSTRACT

BACKGROUND AND OBJECTIVES: Authors of adult rapid response (RRT) studies have established that RRT triggers play an important role in outcomes, but this association is not studied in pediatrics. In this study, we explore the characteristics and outcomes of pediatric rapid response with a respiratory trigger (Resp-RRT). We hypothesize that outcomes differ on the basis of patients' primary diagnoses at the time of Resp-RRT. METHODS: We conducted a 2-year retrospective observational study at an academic tertiary care pediatric hospital. RESULTS: Among the 1287 Resp-RRTs in 1060 patients, those with a respiratory diagnosis (N = 686) were younger, less likely to have complex chronic conditions, and less likely to have concurrent triggers (P < .01) than those with a nonrespiratory diagnosis (N = 601). Patients with a respiratory diagnosis were more likely to receive noninvasive ventilation, less likely to receive vasoactive support, and had lower 30-day mortality (P < .01). Among those with a respiratory diagnosis, the 541 patients with acute illness were younger, less likely to have complex chronic conditions, and less likely to receive vasoactive support than those with acute on chronic illness (N = 100) (P < .01). CONCLUSIONS: Among pediatric respiratory-triggered RRT events, patients with a respiratory diagnosis were more likely to receive acute respiratory support in ICU but have better long-term outcomes. Presence of complex chronic conditions increases risk of acute respiratory support and mortality. The interplay of primary diagnosis with RRT trigger can potentially inform resource needs and outcomes for pediatric Resp-RRTs.


Subject(s)
Hospital Rapid Response Team , Pediatrics , Adult , Child , Humans , Retrospective Studies
3.
4.
Hosp Pediatr ; 8(12): 733-739, 2018 12.
Article in English | MEDLINE | ID: mdl-30385459

ABSTRACT

OBJECTIVES: Asthma exacerbations are a leading cause of hospitalization among children. Despite the existence of hospital protocols and national guidelines, little guidance is available regarding appropriate short-acting ß-agonist (SABA) frequency discharge criteria. Our aim was to reduce the median length of stay (LOS) for children hospitalized with asthma exacerbations by 4 hours by changing the discharge requirement SABA frequency. METHODS: Multiple plan-do-study-act cycles based on findings in our key driver diagram were used to decrease LOS. Our primary intervention was reducing the SABA administration frequency discharge requirement from every 4 hours to every 3 hours. After a feasibility pilot, this change was implemented throughout the hospital. Our intervention bundle included updating our evidence-based guidelines, electronic health record order sets and note templates, house-wide education, and a new process for respiratory therapists to notify physicians of discharge readiness. Our primary metric was LOS, with 3-, 7-, and 14-day same-cause emergency department (ED) revisits and hospital readmissions as balancing metrics. Statistical process control charts and nonparametric testing were performed for data analysis. RESULTS: Median hospital LOS was significantly lower in the postintervention period compared with the preintervention period (30.18 vs 36.14 hours respectively; P < .001). Statistical process control charts indicated special cause variation was achieved. No significant differences were observed in rates of ED revisits or hospital readmissions. CONCLUSIONS: Reducing the discharge requirement of SABA frequency from every 4 hours to every 3 hours resulted in a reduction in LOS, with no increase in ED recidivism or hospital readmission rates.


Subject(s)
Adrenergic beta-2 Receptor Agonists/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Forced Expiratory Volume/drug effects , Patient Discharge/statistics & numerical data , Quality Improvement , Asthma/physiopathology , Child , Child, Preschool , Feasibility Studies , Female , Forced Expiratory Volume/physiology , Humans , Length of Stay/statistics & numerical data , Male , Pilot Projects , Quality Improvement/standards , Treatment Outcome , United States/epidemiology
5.
6.
Case Rep Urol ; 2018: 3479202, 2018.
Article in English | MEDLINE | ID: mdl-30631633

ABSTRACT

Despite the known nephrotoxicity of gentamicin, in 2008 the American Urological Association published guidelines recommending single high-dose weight-based gentamicin prophylaxis of 5 mg/kg for procedures involving urologic prostheses. These guidelines are based on the theoretical renal safety and improved antimicrobial activity of a single large dose of gentamicin. However, the risk of nephrotoxicity after weight-based gentamicin prophylaxis specifically in penile prosthetic surgery has never been established with evidence-based studies. This is of special concern in light of the known high rates of preexisting conditions in this specific population. Therefore, in order to expose potential safety issues, we present three cases of postoperative acute kidney injury following weight-based gentamicin prophylaxis after implantation of inflatable penile prostheses.

7.
Tissue Eng Part C Methods ; 19(9): 665-75, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23301612

ABSTRACT

A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle α-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.


Subject(s)
Bronchioles/cytology , Coculture Techniques/methods , Magnetics , Animals , Cattle , Endothelial Cells/cytology , Endothelial Cells/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Immunohistochemistry , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , Staining and Labeling
9.
J Pediatr ; 160(4): 700-702.e3, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22240110

ABSTRACT

Known genetic causes of pediatric interstitial lung disease include disorders of surfactant metabolism, telomerase, and DNA repair. We report 4 children from 2 families with rapidly progressive and fatal pulmonary fibrosis. A novel DNA repair defect unrelated to the ataxia-telangiectasia mutated gene was found in 1 child from each family.


Subject(s)
DNA Repair-Deficiency Disorders/complications , Pulmonary Fibrosis/genetics , Disease Progression , Humans , Infant, Newborn , Male , Time Factors
11.
Eur J Pharmacol ; 635(1-3): 9-15, 2010 Jun 10.
Article in English | MEDLINE | ID: mdl-20193676

ABSTRACT

The internalization of beta(2)-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on beta(2)-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the beta(2)-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. beta(2)-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A beta(2)-adrenoceptors. The mutant beta(2)-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type beta(2)-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A beta(2)-adrenoceptors was reduced to approximately 25% that of wild type beta(2)-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A beta(2)-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type beta(2)-adrenoceptors. Therefore, although the role of L339,L340 in beta(2)-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient beta(2)-adrenoceptors endocytosis.


Subject(s)
Endocytosis , G-Protein-Coupled Receptor Kinases/metabolism , Leucine/genetics , Leucine/metabolism , Mutant Proteins/metabolism , Mutation , Receptors, Adrenergic, beta-2/metabolism , Amino Acid Motifs/genetics , Animals , Cell Line , Down-Regulation , Humans , Kinetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Phosphorylation/genetics , Protein Transport , Receptors, Adrenergic, beta-2/chemistry , Receptors, Adrenergic, beta-2/genetics , Serine/metabolism
12.
Traffic ; 9(11): 1958-71, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18785920

ABSTRACT

For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.


Subject(s)
Actins/metabolism , Cytoskeleton/metabolism , Myosin Heavy Chains/metabolism , Myosin Type V/metabolism , Receptors, Adrenergic, beta-2/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line , Cytochalasin D/pharmacology , Endocytosis/drug effects , Humans , Kinetics , Thiazolidines/pharmacology
13.
Exp Cell Res ; 313(12): 2586-96, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17553490

ABSTRACT

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.


Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Chromones/pharmacology , Endocytosis/drug effects , Morpholines/pharmacology , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-Agonists/pharmacology , Cell Line , Green Fluorescent Proteins/metabolism , Humans , Protein Transport/drug effects , Transferrin/metabolism , rab GTP-Binding Proteins/metabolism , rab7 GTP-Binding Proteins
14.
Mol Pharmacol ; 71(1): 47-60, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17012621

ABSTRACT

Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the beta2-adrenergic receptor (beta2AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t(1/2) of 9 min (k = 0.08 +/- 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 microM isoproterenol for 5 min exhibited a lag phase of approximately 5 min, after which dephosphorylation proceeded slowly with a t(1/2) of 18 min (k = 0.039 +/- 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated beta2AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated beta2AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 +/- 0.009/min; t(1/2) = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.


Subject(s)
Receptors, Adrenergic, beta-2/metabolism , Adenylyl Cyclases/metabolism , Cell Line , Cell Membrane/enzymology , Cyclic AMP-Dependent Protein Kinases/metabolism , G-Protein-Coupled Receptor Kinase 1/metabolism , Humans , Isoproterenol/pharmacology , Kidney , Kinetics , Phosphoserine/metabolism , Receptors, Adrenergic, beta-2/drug effects
15.
Respir Med ; 101(5): 1007-14, 2007 May.
Article in English | MEDLINE | ID: mdl-17052901

ABSTRACT

BACKGROUND: Current guidelines recommend repeated doses of albuterol for the emergency treatment of acute asthma. However, approximately one-third of patients show little or no initial response to this partial beta(2)-agonist. METHODS: We conducted a randomized, double-blind, proof-of-concept study to investigate whether a full beta(2)-agonist, isoproterenol, offers a therapeutic advantage in adults presenting with acute severe asthma (FEV(1)<50%) who fail to respond to an initial treatment of the partial beta(2)-agonist, albuterol. Study subjects were randomized to receive a 2-h continuous nebulization of either albuterol (7.5mg/h) (n=10, mean FEV(1)=37% predicted) or isoproterenol (7.5mg/h) (n=9, mean FEV(1)=33% predicted). Respiratory symptoms, vital signs and pulmonary function measures were collected. RESULTS: Subjects from both treatment groups had similar baseline characteristics. The percent improvements from baseline FEV(1) at 60 and 120min were significantly higher in subjects receiving isoproterenol than those receiving albuterol (44 vs. 17% and 63 vs. 24%, respectively, P<0.05). The change in symptoms measured by the modified Borg score was also significantly greater in subjects receiving isoproterenol (P<0.01). Both treatments were well tolerated, though the mean increase in pulse rate at 60 and 120min (21 vs. 1 and 23 vs. 6beats/min, respectively, P<0.05) and the mean change in serum potassium at 120min (-0.52 vs. -0.07meq/L, P<0.05) from baseline were significantly greater in the isoproterenol group. CONCLUSIONS: Our data suggest that in subjects presenting with acute severe asthma who fail to show an initial response to albuterol, the use of a beta(2)-agonist of higher intrinsic efficacy can be more effective in improving lung function and symptoms.


Subject(s)
Adrenergic beta-Agonists/therapeutic use , Albuterol/therapeutic use , Asthma/drug therapy , Bronchodilator Agents/therapeutic use , Isoproterenol/therapeutic use , Acute Disease , Adolescent , Adrenergic beta-Agonists/adverse effects , Adult , Albuterol/adverse effects , Asthma/physiopathology , Blood Pressure/drug effects , Bronchodilator Agents/adverse effects , Double-Blind Method , Female , Forced Expiratory Volume/drug effects , Heart Rate/drug effects , Humans , Isoproterenol/adverse effects , Male , Middle Aged , Treatment Failure , Treatment Outcome
16.
Am J Respir Cell Mol Biol ; 36(2): 254-61, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16980556

ABSTRACT

Salmeterol is a long-acting beta(2)-adrenergic receptor (beta(2)AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other beta-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting beta-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of beta-agonists. This effect may not be the same with all beta-agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high-affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, down-regulation, and internalization difficult. We determined the capacity of salmeterol to induce beta(2)AR endocytosis, G protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and beta-arrestin2 translocation in HEK293 cells as compared with other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy, such as epinephrine and formoterol, salmeterol did not induce significant beta(2)AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-beta-arrestin2 chimera (EGFP-beta-arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-beta-arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit beta-arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.


Subject(s)
Albuterol/analogs & derivatives , Endocytosis/drug effects , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-2 Receptor Agonists , Adrenergic beta-Agonists/pharmacology , Albuterol/pharmacology , Arrestins/metabolism , Enzyme-Linked Immunosorbent Assay , Epinephrine/pharmacology , Fluorescent Antibody Technique , Gene Expression/drug effects , Humans , Phosphorylation/drug effects , Protein Transport/drug effects , Receptors, Adrenergic, beta-2/analysis , Salmeterol Xinafoate , beta-Adrenergic Receptor Kinases/metabolism
17.
Pediatrics ; 118(3): e924-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16923925

ABSTRACT

Here we present the unusual case of an adolescent with cystic fibrosis presenting with declining pulmonary function and diffuse micronodular pulmonary disease. This case illustrates the radiographic and pathologic findings associated with the intravenous injection and pulmonary arterial embolization of insoluble pharmaceutical-tablet constituents. The number of first-time users reporting nonmedical use of prescription pain relievers is increasing dramatically, especially in adolescents. Recognition of both the diagnostic imaging features and histologic features on lung biopsy are critical steps for early diagnosis, intervention, and potential prevention of sudden death in these at-risk patients.


Subject(s)
Cystic Fibrosis , Embolism/etiology , Foreign-Body Reaction , Solitary Pulmonary Nodule/diagnosis , Substance-Related Disorders/diagnosis , Tablets/adverse effects , Acetaminophen/administration & dosage , Acetaminophen/adverse effects , Adolescent , Diagnosis, Differential , Drug Combinations , Excipients/administration & dosage , Excipients/adverse effects , Fatal Outcome , Female , Humans , Hydrocodone/administration & dosage , Hydrocodone/adverse effects , Injections, Intravenous , Pulmonary Artery/pathology , Respiratory Function Tests , Tablets/administration & dosage
18.
J Biol Chem ; 281(11): 7684-92, 2006 Mar 17.
Article in English | MEDLINE | ID: mdl-16407241

ABSTRACT

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.


Subject(s)
Arrestins/chemistry , G-Protein-Coupled Receptor Kinase 1/chemistry , Receptors, Adrenergic, beta-2/metabolism , Adenylyl Cyclases/metabolism , Alanine/chemistry , Aspartic Acid/chemistry , Binding Sites , Blotting, Western , Cell Line , Cell Membrane/metabolism , Cyclic AMP-Dependent Protein Kinases/chemistry , DNA, Complementary/metabolism , Epinephrine/pharmacology , G-Protein-Coupled Receptor Kinase 1/metabolism , Green Fluorescent Proteins/metabolism , Humans , Isoproterenol/chemistry , Kinetics , Mutation , Phosphorylation , Pindolol/analogs & derivatives , Pindolol/pharmacology , Protein Structure, Tertiary , Protein Transport , Serine/chemistry , Time Factors , Transfection , beta-Arrestin 1 , beta-Arrestin 2 , beta-Arrestins
19.
Proc Natl Acad Sci U S A ; 102(13): 4854-9, 2005 Mar 29.
Article in English | MEDLINE | ID: mdl-15781872

ABSTRACT

Misfolding and aggregation of proteins play an important part in the pathogenesis of several genetic and degenerative diseases. Recent evidence suggests that cells have evolved a pathway that involves sequestration of aggregated proteins into specialized "holding stations" called aggresomes. Here we show that cells regulate inducible NO synthase (iNOS), an important host defense protein, through aggresome formation. iNOS aggresome formation depends on a functional dynein motor and the integrity of the microtubules. The iNOS aggresome represents a "physiologic aggresome" and thus defines a new paradigm for cellular regulation of protein processing. This study indicates that aggresome formation in response to misfolded proteins may merely represent an acceleration of an established physiologic regulatory process for specific proteins whose regulation by aggresome formation is deemed necessary by the cell.


Subject(s)
Inclusion Bodies/metabolism , Nitric Oxide Synthase/metabolism , Animals , Cells, Cultured , Dyneins/metabolism , Fluorescence Recovery After Photobleaching , Green Fluorescent Proteins , Humans , Image Processing, Computer-Assisted , Inclusion Bodies/drug effects , Indoles , Leupeptins/pharmacology , Mice , Microscopy, Electron , Microtubule-Organizing Center/drug effects , Microtubule-Organizing Center/metabolism , Nitric Oxide Synthase Type II , Nocodazole/pharmacology
20.
Curr Drug Targets Inflamm Allergy ; 3(3): 271-7, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15379595

ABSTRACT

Beta2-adrenergic agonists (beta2-agonists) play a pivotal role in the acute and chronic management of asthma. Their major action on the airways is the relaxation of smooth muscle cells. In addition to their bronchodilator properties, beta2-agonists may have other effects through their activation of beta2-receptors expressed on resident airway cells such as epithelial cells and mast cells and circulating inflammatory cells such as eosinophils and neutrophils. These non-bronchodilator activities of beta2-agonists may enhance their efficacy in the management of asthma. In pre-clinical studies, the anti-inflammatory effects of beta2-agonists are demonstrated through their stabilizing effect on mast cells and their inhibition of mediator release from eosinophils, macrophages T-lymphocytes, and neutrophils. In addition, beta2-agonists may inhibit plasma exudation in the airway, the release of neuropeptides from sensory nerves, and mediator release from epithelial cells. These in vitro observations are not as clearly demonstrated in clinical trials, which may be explained by the rapid desensitization of beta2-adrenergic receptors on airway inflammatory cells. The regular use of short-acting beta2-agonists alone has been shown to have deleterious effects on asthma control. Therefore, short-acting agents should only be used when needed for rescue of acute symptoms. Monotherapy with long-acting beta2-agonists has also been associated with poor asthma control. However, when given concomitantly with inhaled corticosteroids, beta2-agonists may potentiate the anti-inflammatory effect of corticosteroids, improve asthma control and prevent exacerbations.


Subject(s)
Adrenergic beta-2 Receptor Agonists , Anti-Inflammatory Agents/pharmacology , Asthma/drug therapy , Adrenal Cortex Hormones/therapeutic use , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Clinical Trials as Topic , Drug Therapy, Combination , Genetic Variation , Humans , Receptors, Adrenergic, beta-2/genetics , Receptors, Glucocorticoid/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...