Measurements of cytosolic concentration of cAMP and L-lactate in cultured rat astrocytes and 3T3 cells

Z G-proteini sklopljeni receptorji (GPCR) so transmembranski proteini med katere spada več kot 800 receptorjev. Pri ljudeh je ena tretjina le-teh tarča odobrenih zdravil. V tem delu smo raziskali GPRB, GPCR sirota, ki spada v družino super ohranjenih receptorjev, izraženih v možganih, z neznano funkcijo. Citosolne ravni L-laktata, končnega produkta aerobne glikolize, smo merili z nanosenzorjem Laconic, ki temelji na metodi FRET. V posameznih embrionalnih celicah divjega tipa (WT) 3T3 je uporaba agonistov Smart009 in Smart075, ter molekule 8535 (1 µM), nadomestnega agonista, za katerega je znano, da aktivira GPRB, povzročila povečanje koncentracije L-laktata. Podobno povečanje je bilo zabeleženo v primarnih astrocitih podgan, nevroglialnih celic, ki vsebujejo glikogen in izražajo encime aerobne glikolize. Pri celicah 3T3, ki niso izražale GPRB (3T3KOB), je bilo povečanje L-laktata, ki ga povzročata agonista Smart009 in 8535, zmanjšano v primerjavi s kontrolami WT. V celicah 3T3KOB smo povečanje L-laktata po stimulaciji ponovno vzpostavili s pomočjo plazmida, ki nosi GPRB. Ti rezultati kažejo, da stimulacija GPR27 poveča aerobno glikolizo in proizvodnjo L-laktata v celicah 3T3 in astrocitih. Spremljali smo tudi podcelično dinamiko L-laktata in cAMP in ugotovili, da stimulacija z agonisti za GPCR izzove prostorsko heterogene spremembe v znotrajcelični porazdelitvi L-laktata in cAMP.G-protein coupled receptors (GPCRs) are transmembrane proteins that include more than 800 receptors. One third of these are the target of approved drugs in humans. In this work, we investigated GPRB, an orphan GPCR belonging to a family of super-preserved receptors expressed in the brain with unknown function. The cytosolic levels of L-lactate, the end product of aerobic glycolysis, were measured with a Laconic nanosensor based on the FRET method. In individual wild-type (WT) 3T3 embryonic cells, the use of the agonists Smart009 and Smart075, and molecule 8535 (1 µM), a replacement agonist known to activate GPRB, resulted in an increase in L-lactate concentration. A similar increase was observed in primary rat astrocytes, glycogen-containing neuroglial cells expressing aerobic glycolysis enzymes. In 3T3 cells that did not express GPRB (3T3KOB), the increase in [lactate]i caused by the Smart009 and 8535i agonists was reduced compared to WT controls. In 3T3KOB cells, the increase in L-lactate was restored after stimulation with a GPRB-expressing plasmid. These results suggest that GPR27 stimulation increases aerobic glycolysis and L-lactate production in 3T3 cells and astrocytes. We also monitored the subcellular dynamics of L-lactate and cAMP and found that stimulation with GPCR agonists induces spatially heterogeneous changes in the intracellular distribution of L-lactate and cAMP