Figure 5. Norepinephrine requires OCT transporters but not receptor internalization to stimulated PI4P hydrolysis.

NRVMs were transduced with FAPP-PH-GFP and stimulated with norepinephrine (10 μM) in the presence of corticosterone (100 μM, (A), abacavir (10 μM, (B), lamotrigine (10 μM, (C) or Dyngo (40 μM, (D). Data are not significant between norepinephrine and norepinephrine + Dyngo. Images were collected as in Figure 1A for PI4P hydrolysis (N = 3) were from at least 4 cells each from separate preparations of NRVMs. Agonists were added where indicated by the arrow. (E) Dyngo has no effect on NES-Venus-mini-Gs Golgi recruitment by norepinephrine. Representative image of norepinephrine-mediated NES-Venus-mini-Gs recruitment in the presence of Dyngo (40 μM). (N = 3) Scale bars are 10 μm. (F) NRVMs were transduced with adenovirus containing FRB-CFP-Nb80 and FKBP-GalT-mApple along with adenovirus containing FAPP-PH-GFP for 24 hr prior to experimentation. NRVMs were incubated with either rapamycin (1 μM) or DMSO control for 15 min prior to addition of NE (10 μM) added at the arrow. Images were collected as in Figure 1A for PI4P hydrolysis and were from at least n = 10 cells each from three separate preparations of NRVMs. Data were analyzed as means from N = 3 experiments.

Figure 5—figure supplement 1. Receptor internalization does not contribute to Golgi β1AR localization.

(A) Dyngo blocks Isoproterenol induced β1-AR internalization. NRVMs were transfected with FLAG-β1-AR receptors and allowed to express for 48 hr. Cells were then stimulated with either vehicle control, Isoproterenol (10 μM), dobutamine (100 nM) or norepinephrine (10 μM) for 30 mins. Pretreatment with Dyngo (40 μM) was performed for 15 mins before agonist addition, where indicated. Cells are pseudocolored Green for M2-FLAG-Cy3 antibody, which is bound to FLAG-β1-AR and nuclei are stained with DAPI. Images are representative from at least three experiments. Scale bars are 10 μM. (B) Corticosterone has no effect on the ability of plasma membrane β1-AR to signal. NRVMs were stimulated in the presence of either Corticosterone (100 μM) or vehicle control with the indicated agonists for 10 mins. Cells were then lysed and cAMP measured according to the manufacturer’s instructions. Data is from three experiments. (C) Corticosterone does not inhibit Dobutamine-mediated PI4P hydrolysis. NRVMs were transduced with FAPP-PH-GFP and stimulated with dobutamine (100 mM) in the presence of Corticosterone (100 μM) or vehicle and analyzed as in Figure 1A. Images for PI4P hydrolysis collected as in Figure 1A, were from at least n = 7 cells each from three separate preparations of NRVMs. Agonists were added where indicated by the arrow.

Figure 5—video 1. Inhibition of receptor internalization does not alter activation of β1ARs in the Golgi apparatus.

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DOI: 10.7554/eLife.48167.021

Confocal images of β1-ARs-overexpressing cardiomyocytes with NES-Venus-mini-Gs, pretreated with 40 µM Dyngo. Total time represented by the movie is 30 min and pictures were taken every 30 s. Dyngo was added 15 min prior to experimentation and norepinephrine was added at 2 min timepoint.