PubMed Health⌕ Search

Biomedical subjects

R B Penn

Publications and source records attributed to R B Penn.

29 records · Page 2Linked to original sources
PubMed1996

Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor.

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.

Animals↗
PubMed1996

Effects of inflammation and acute beta-agonist inhalation on beta 2-AR signaling in human airways.

Although alterations in beta 2-adrenergic receptor (AR) responsiveness may in part explain reports linking deterioration of asthma control with beta-agonist treatment of asthmatics, few data exist on beta 2-AR regulation in human airway cells. We have employed a bronchoscopy model to examine inflammation- and beta-agonist-induced alterations in human bronchial epithelial cell beta 2-AR density and responsiveness. Allergic asthmatic subjects participated in 2-day protocols examining airways before and 24 h after segmental antigen challenge (SAC) with ragweed. To assess the effect of acute beta-agonist exposure, bronchoscopies were performed both with (+ beta-Ag) and without (-beta-Ag) inhalation of beta-agonist 30 min before the procedure. Measurements of inflammatory cell infiltration were obtained by analysis of bronchoalveolar lavage fluid, and beta 2-AR density and responsiveness were examined in bronchial epithelial cells obtained by bronchoscopic brushing. Neither SAC nor acute beta-agonist administration alone significantly affected epithelial cell beta 2-AR density. beta-Agonist-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) generation was significantly lower in the + beta-Ag groups compared with the-beta-Ag group, demonstrating acute agonist-specific beta 2-AR desensitization in vivo. SAC caused a small, statistically insignificant reduction in beta-Agonist-stimulated cAMP production in both -beta-Ag or + beta-Ag groups. These lata suggest that acute beta-agonist inhalation, but not airway inflammation, significantly reduces maximal beta 2-AR responsiveness in airway cells.

Albuterol↗
PubMed1994

Structure of the human gene encoding the beta-adrenergic receptor kinase.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied forms of the beta 2-adrenergic receptor and related G protein-coupled receptors. beta ARK is one of the best characterized members of a growing family of G protein-coupled receptor kinases. In this article we report the isolation and structural organization of the human beta ARK gene. The gene spans approximately 23 kilobases and is composed of 21 exons interrupted by 20 introns. Exon sizes range from 52 bases (exon 7) to over 1200 bases (exon 21), intron sizes from 68 bases (intron L) to 10.8 kilobases (intron A). The splice sites for donor and acceptor were in agreement with the canonical GT/AG rule. Functional regions of beta ARK are described with respect to their location within the exon-intron organization of the gene. Primer extension and RNase protection assays suggest a major transcription start site approximately 246 bases upstream of the start ATG. Sequence analysis of the 5'-flanking/promoter region reveals many features characteristic of mammalian housekeeping genes, i.e. the lack of a TATA box, an absent or nonstandard positioned CAAT box, high GC content, and the presence of Sp1-binding sites. The extraordinarily high GC content of the 5'-flanking region (> 80%) helps define this region as a CpG island that may be a principal regulator of beta ARK expression.

Amino Acid Sequence↗
PubMed1994

Regulation of beta-agonist- and prostaglandin E2-mediated adenylyl cyclase activity in human airway epithelial cells.

Despite the importance of beta 2-adrenergic receptor (beta 2AR) stimulation in mediating airway epithelial cell function, little is known regarding its regulation in airway epithelium. Perturbations of the airway environment associated with disease states, including the management of bronchomotor tone with beta-agonists, expose airways to putative regulators of beta 2AR signal transduction. In this communication, we describe the desensitization of beta 2AR signal transduction in the human airway epithelial cell line BEAS-2B. Examination of both beta-agonist- and prostaglandin E2 (PGE2)-mediated cAMP generation in BEAS-2B cells reveals both agonist-specific (homologous) and non-agonist-specific (heterologous) desensitization of these G protein-coupled receptor pathways. Short-term homologous desensitization of beta 2AR-mediated cAMP generation was characterized by an approximately 60% loss of maximal responsiveness to isoproterenol (ISO) when cells were pretreated 30 min with 10 microM ISO. A reduced sensitivity to ISO was also evidenced by an approximately 4-fold increase in the EC50 for ISO stimulation of adenylyl cyclase (AC). Short-term heterologous desensitization was characterized by an increase in EC50 (approximately 2- to 3-fold) with no change in maximal responsiveness to ISO in cells pretreated with either forskolin or PGE2. Qualitatively similar findings characterized short-term homologous and heterologous desensitization of PGE2-mediated AC activity. Short-term agonist-specific desensitization of the beta 2AR was associated with, but not dependent upon, rapid beta 2AR sequestration. Long-term pretreatment of cells with 10 nM ISO and 1 microM PGE2 eliminated AC responsiveness to subsequent ISO and PGE2 stimulation, respectively. Exposure of BEAS-2B cells to ISO for 24 h resulted in an approximately 70% loss of beta 2ARs, whereas chronic forskolin or PGE2 pretreatment had no effect on beta 2AR number. Long-term pretreatment of cells designed to elicit heterologous desensitization was associated with reductions in maximal responsiveness to ISO and PGE2 that appear to be related to a loss in inherent AC activity. These findings hold strong implications regarding the effect of beta 2AR desensitization on epithelial cell function and the role of beta-agonists in the management of airway disease.

Adenylyl Cyclases↗
PubMed1989

In vivo mechanical properties of the developing airway.

The inherent mechanical characteristics of the airways are determined in part by their elastic and viscoelastic properties. As compliant structures during early development, the airways are susceptible to significant distention and collapse, depending on the proportionality between airway volume and transmural pressure. To characterize the age-related changes in airway mechanical properties, the elastic and viscoelastic behavior of in vivo tracheal segments were evaluated in preterm and newborn lambs over a wide range of developmental age (108 to 154 days postconceptional age). Tracheal pressure-vol relationships and concomitant airway compliance measurements were used to determine elastic behavior. Calculations of the tracheal relaxation time constant on the same tracheal segments were used to evaluate airway viscoelastic behavior. Data demonstrated a significant (p less than 0.01) correlation with developmental age. With increasing age, the airways were found to be less compliant, and the tracheal relaxation time constant was observed to decrease. The difference in elastic properties of the trachea, in vivo compared to in vitro, suggest that neural-humoral and surrounding connective tissue factors may affect the elasticity of the developing airway. Although the modulating effects of smooth muscle tone and supporting connective tissue assist in the control of airway dimension and resistance to airflow in the intact airway, the age-related differences in the elastic properties may be a factor that predisposes the more immature airway to positive pressure-induced damage.

Animals↗
PubMed1989

Developmental differences in tracheal cartilage mechanics.

Length-tension characteristics were examined in tracheal rings excised from preterm lambs (n = 8) and adult sheep (n = 7) to determine developmental differences in the mechanical properties of tracheal cartilage. Tension was measured in both the complex (comprised of the pars membranacea and its cartilagenous insertion) and the cartilage alone (by severing the pars membranacea) as the ring was lengthened so as to increase the distance between the points of insertion. Analysis of regression lines show significantly greater slopes for both the complex and the cartilage in the adult group compared to the preterm group. These data reveal that age-related differences in tracheal cartilage mechanics parallel and may contribute to age-related differences previously determined in tracheal compliance.

Age Factors↗
PubMed1988

Influence of smooth muscle tone and longitudinal tension on the collapsibility of immature airways.

Mechanical properties and pressure-flow relationships of tracheae excised from very premature lambs were studied in a plethysmograph. Control (Group I) data revealed the tracheae to be extremely compliant, collapsible airways, with an inflation compliance (Si) of 0.033 (+/- 0.004 SE) mmHg-1, collapsing compliance (Sc) of 0.026 (+/- 0.001 SE) mmHg-1, and pressure-flow relationships similar to those of a Starling resistor. Acetylcholine administration (Group II) lowered both Si, 0.026 (+/- 0.003 SE) mmHg-1 and Sc, 0.022 (+/- SE) mmHg-1, as did longitudinal stretch (Group III): Si, 0.021 (+/- .003 SE) mmHg-1, and Sc, 0.017 (+/- 0.002 SE) mmHg-1. Alterations in tracheal collapsibility were also evidenced by significant reductions in resistance to airflow when the tracheae were subjected to compressive forces. Altering both smooth muscle tone (acetylcholine administration) and longitudinal length simultaneously (Group IV) produced results similar to those obtained for Group III. These data help to define the functional characteristics of immature airways and may provide insight for more effective clinical management of the premature infant.

Acetylcholine↗
PubMed1988

Effect of tracheal smooth muscle tone on collapsibility of immature airways.

To test the influence of smooth muscle tone on extremely immature airways, tracheal segments (n = 19) were excised from premature lambs at 114-121 days gestation and mounted in a chamber filled with Krebs solution. Inflation (Si) and collapsing (Sc) compliance were determined by altering transmural pressure from 30 to 0 Torr and -30 to 0 Torr, respectively, both during control (C) and after acetylcholine (ACh) administration (experimental, E). Flow (V = 2-15 l/min) was then introduced through the tracheal lumen while chamber pressure (Pc) was increased from 0 to 30 Torr and driving pressure (Pd) was recorded for both C and E conditions. Tracheae were found to be extremely compliant; both Si and Sc were significantly (P less than 0.005) lower after ACh administration. Resistance to airflow (R = Pd/V) was also significantly (P less than 0.05) lower after ACh administration at each compressive pressure and each flow value. These results suggest that the highly compliant preterm trachea exhibits pressure-flow characteristics similar to a Starling resistor, and the effects of compressive pressures may be attenuated by ACh-induced smooth muscle contraction. Comparison of these results with data from adult and newborn animals suggests a developmental difference in tracheal mechanics and pressure-flow relationships, as well as in the way airway function is altered by smooth muscle stimulation.

Acetylcholine↗
PubMed1988

Effect of ventilation on mechanical properties and pressure-flow relationships of immature airways.

Ventilation of immature airways has been shown to result in pressure-induced deformation and alteration of mechanical properties. These changes in mechanical properties may alter the effect of compressive pressures on pressure-flow relationships. To test this hypothesis, unventilated (Group I, n = 8) and ventilated (group II, n = 8; mean pressures of 8-12 cm H2O for 2 h duration) tracheal segments were excised from preterm lambs approximately 118 days gestation and mounted in a Krebs-filled chamber for determination of compliances and pressure-flow relationships. Compliance data were obtained for both the inflation loop (inflation compliance) and the collapsing loop (collapsing compliance) of the pressure-volume curve for each segment. Flow (V = 2-10 liter/min.) was introduced through the lumen of each segment at different chamber pressures (Pc = 5-30 mm Hg). Driving pressure (Pd) was recorded for each V and Pc. Airway resistance (R) was calculated as Pd/V. Group II had significantly greater (p less than 0.005) radii than Group I, suggesting ventilation-induced deformation. Group II had significantly lower (p less than 0.05) inflation compliance but significantly greater (p less than 0.05) collapsing compliance than group I. Two-factor analysis of variance demonstrated significantly greater (p less than 0.05) R in group II at each value of V and each Pc. These data may help to explain the flow limitation and gas-trapping observed in preterm infants who have been mechanically ventilated.

Animals↗
PubMedDate not supplied

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

Animals↗