Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Novel inhibition of proteoglycan synthesis and exocytosis by diethylcarbamazine in the Swarm rat chondrocyte

Pretreatment of cultured chondrosarcoma chondrocytes at 37 degrees C for 15 min with 15 mM diethylcarbamazine (DEC) followed by a 60-min pulse with [35S] sulfate in the presence of DEC resulted in an approximate 40% inhibition of synthesis and a 75% inhibition of secretion of 35S-proteoglycan. The inhibition was dose-related and was not due to a decrease in protein synthesis. Chondrocytes exposed for 75 min to 15 mM DEC, washed, incubated for 17 h in DEC-free medium, and then pulsed with [35S]sulfate showed no inhibition in the rate of synthesis of proteoglycan or in the per cent of radiolabeled proteoglycans exocytosed into the culture medium, indicating full reversibility of the inhibitory effect. When chondrocytes were incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, secretion of beta-D-xyloside-bound 35S-glycosaminoglycan was inhibited by more than 70% despite an approximate 3-fold increase in intracellular 35S-macromolecules, as compared to cells exposed to beta-D-xyloside alone. Upon removal of DEC, the block in the secretion of beta-D-xyloside-bound 35S-glycosaminoglycans was reversed, although there was a 15-30-min lag in the initiation of exocytosis. Light and electron microscopic examination of chondrocytes after 75 min of incubation with 15 mM DEC revealed large vacuoles, a distended Golgi apparatus, and a distended endoplasmic reticulum which contained electron dense material. Upon removal of DEC, the vacuoles disappeared and distended organelles returned to their normal appearance between 15 and 30 min, coincident with the start of exocytosis of 35S-proteoglycan and beta-D-xyloside-bound 35S-glycosaminoglycan. These biochemical and morphological studies indicate that DEC treatment of chondrosarcoma chondrocytes alters the transport of molecules from the endoplasmic reticulum to the Golgi and the transport of molecules from the Golgi to the cell surface.     

Initial decomposition ofNymphoides peltata (Gmel.) O. Kuntze (Menyanthaceae), as studied by the leaf-marking method

Summary In this paper the leaf-marking method as used for the study of the development and initial decomposition of floating leaves is described and the reliability of the various measurements is tested and/or discussed. Some general results obtained withNymphoides peltata (Gmel.) O. Kuntze in tanks and in the field are presented and crltically discussed. Autolysis followed by microbial decay was in all cases the most important factor by which leaves disintegrated. In the field plots animals were responsible for the disappearance of 22% of the total leaf area produced during a growth season. This is, however, the combined effect of consumption and damage succeeded by microbial decay. Real grazing can be estimated to be no more than 10% of the production of floating leaves. Fungi can have an important role in initial decomposition, especially after the flowering period, as is demonstrated forSeptoria villarsiae Desm. All damage types show temporal and, in the case of animals, also spatial distribution patterns.     

Phosphatidylinositol and phosphatidic acid metabolism in rat pancreatic islets in response to neurotransmitter and hormonal stimuli

Carbamylcholine produced a concentration-dependent stimulation of labelling of phosphatidylinositol and phosphatidic acid in rat islets of Langerhans following preincubation with 32PO43(-). The time course of these effects suggested that the initial action of carbamylcholine was to stimulate phosphatidic acid production, presumably by causing hydrolysis of phosphatidylinositol. This conclusion was substantiated by experiments in which islet phospholipids were pre-labelled with [3H]arachidonic acid. Under these conditions, carbamylcholine caused a loss of radioactivity from phosphatidylinositol, together with an increase in labelling of phosphatidic acid. The effects of carbamylcholine on islet phospholipid labelling were not dependent upon the presence of added Ca2+, but were abolished by EDTA and by atropine. An apparent stimulation of phosphatidylinositol and phosphatidic acid metabolism was also induced by cholecystokinin-pancreozymin, whereas glucagon, arginine, glibenclamide and thyrotropin had no significant effect. The data suggest that enhanced activity of the so-called phosphatidylinositol cycle may be an important event in regulating secretory activity of islets in response to certain neurotransmitter and hormonal stimuli. Furthermore, the results are compatible with the hypothesis that increased phospholipid metabolism may play a role in the modulation of ionic fluxes during stimulation by such agents.     

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

Cytotoxicity of cholesterol oxidase to cells of hypercholesterolemic guinea pigs

1. A high cholesterol diet caused guinea pig erythrocytes to become sensitive to lysis by cholesterol oxidase (CO), a protein not hemolytic to normal cells. 2. Lysis was associated with conversion of membrane cholesterol to its oxidation product (delta-4-cholesten-3-one). 3. Intravenous injection of CO to hypercholesterolemic guinea pigs produced a reduction in serum cholesterol, but was not lethal as it was in rabbits. 4. Homogenized spleen, liver and kidney from the hyperlipidemic animals were sensitive to in vitro cholesterol oxidation while tissues from non-lipemic animals were resistant to modification.