Competing G protein‐coupled receptor kinases balance G protein and β‐arrestin signaling

Seven‐transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β‐arrestins, whose recruitment to the activated receptor is regulated by G protein‐coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal‐regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT_1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)‐based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well‐established function in the desensitization of G‐protein activation, GRK2 exerts a strong negative effect on β‐arrestin‐dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2‐dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT_1AR, and HEK293 cells expressing other 7TMRs.     

Effect of dietary docosahexaenoic acid on biosynthesis of docosahexaenoic acid from alpha-linolenic acid in young rats

Docosahexaenoic acid (DHA), a crucial nervous system n-3 PUFA, may be obtained in the diet or synthesized in vivo from dietary alpha-linolenic acid (LNA). We addressed whether DHA synthesis is regulated by the availability of dietary DHA in artificially reared rat pups, during p8 to p28 development. Over 20 days, one group of rat pups was continuously fed deuterium-labeled LNA (d5-LNA) and no other n-3 PUFA (d5-LNA diet), and a second group of rat pups was fed a d5-LNA diet with unlabeled DHA (d5-LNA + DHA diet). The rat pups were then euthanized, and the total amount of deuterium-labeled docosahexaenoic acid (d5-DHA) (synthesized DHA) as well as other n-3 fatty acids present in various body tissues, was quantified. In the d5-LNA + DHA group, the presence of dietary DHA led to a marked decrease (3- to 5-fold) in the total amount of d5-DHA that accumulated in all tissues that we examined, except in adipose. Overall, DHA accretion from d5-DHA was generally diminished by availability of dietary preformed DHA, inasmuch as this was found to be the predominant source of tissue DHA. When preformed DHA was unavailable, d5-DHA and unlabeled DHA were preferentially accreted in some tissues along with a net loss of unlabeled DHA from other organs.     

Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.     

Metal accumulation in two contiguous eutrophic peri-urban lakes,Chivero and Manyame,Zimbabwe

Concentrations of aluminium, cadmium, chromium, cobalt, copper, iron, lead, nickel and zinc were determined in surface water, benthic sediments, and the gills, liver and stomach muscle tissues of Oreochromis niloticus and Clarias gariepinus in peri-urban lakes Chivero and Manyame, Zimbabwe. Five sites were sampled in each lake once per month in November 2015, February, May, August and November 2016. Pollution load index detected no metal contamination, whereas the geo-accumulation index reflected heavy to extreme sediment pollution, with Fe, Cd, Zn, Cr, Ni and Cu present in both lakes. Significant spatial temporal variations were detected for Al, Cr, Cu and Pb across sites within and between the two lakes. High Fe, Al and Cr concentrations in water and sediments in lakes Chivero and Manyame derive from geogenic background sources in addition to anthropogenic loads and intensity. Elevated concentrations of Al, Pb, Cu, Cd, Fe and Zn detected in gills, liver and stomach tissue of catfish corroborate concentrations in water and sediments, and pose the highest ecological and health risk for hydrobionts in lakes Chivero and Manyame. Contiguity of peri-urban lakes exposes them to similar threats, necessitating creative water management strategies, which ensure ecological continuity.