Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: siRNA silencing of PDE3A (siPDE3A) blunted NO‐induced AMPK activation. Human PASMC were transfected with siPDE3A, siPDE3B, or scramble siRNA. After 48 hr, cells were treated with DETA NONOate (250 μM) for 24 hr. Protein was harvested, analyzed by Western blot, and expression quantified by densitometry. (a) Representative Western blots for each PDE3A, PDE3B, β‐actin, and p‐ and total AMPK. Data are shown as fold changes ± SEM relative to scramble siRNA control for (b) PDE3A/β‐actin after siPDE3A or siPDE3B transfection; n = 5–6 for each group, * p < .025 different from scramble siRNA (two‐way ANOVA). (c) PDE3B/β‐actin after siPDE3A or siPDE3B transfection; n = 6 for each group, * p < .005 different from scramble siRNA (two‐way ANOVA), and (d) p/T‐AMPK after siPDE3A or siPDE3B transfection; n = 5–6 for each group, * p < .02 different from scramble siRNA, control; # p < .001 different from scramble siRNA, DETA NONOate; & p < .003 different from siPDE3A, DETA NONOate (two‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Transfection, Western Blot, Expressing, Control

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: sGC stimulator increased PDE3A protein expression and AMPK phosphorylation, whereas sGC inhibitor prevented the NO‐induced increase in PDE3A protein expression and AMPK phosphorylation in hPASMC. Human PASMC were treated with the NO donor DETA NONOate (250 μM) or sGC stimulator, BAY 41‐2272 (10 μM). Protein was harvested, analyzed by Western blot, and expression quantified by densitometry. Representative Western blots with data shown below as fold changes ± SEM relative to vehicle control for (a) PDE3A and β‐actin; n = 6–11 for each group, * p < .04 different from control (one‐way ANOVA) and (b) p‐AMPK and total AMPK; n = 6–9 for each group, * p < .03 different from control (one‐way ANOVA)l. Alternatively, hPASMC were pretreated for 4 hr with the sGC inhibitor, ODQ (10 μM), then incubated with DETA NONOate (250 μM) for 48 hr. Representative Western blots with data below shown as fold changes ± SEM relative to vehicle control for (c) PDE3A and β‐actin; n = 8–11 for each group, * p < .001 different from control, # p < .001 different from DETA NONOate (one‐way ANOVA) and (d) p‐AMPK and total AMPK; n = 8–9 for each group, * p < .001 different from control, # p < .001 different from DETA NONOate (one‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Incubation

Journal: Physiological Reports

Article Title: Nitric oxide activates AMPK by modulating PDE3A in human pulmonary artery smooth muscle cells

doi: 10.14814/phy2.14559

Figure Lengend Snippet: Treatment with NO, AMPK agonist, and PDE3 inhibitor decreased hPASMC proliferation. Human PASMC were treated with DETA NONOate (250 μM), the AMPK agonist, AICAR (5 μM), the PDE3 inhibitor, milrinone (10 μM), the combination of DETA NONOate + milrinone, or vehicle and incubated for 48 hr. MTT proliferation assay was performed. Data shown are means ± SEM . n = 6–10, * p < .001 different from control, # p < .003 different from AICAR (one‐way ANOVA)

Article Snippet: AICAR (5‐Aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside), an AMPK agonist, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Incubation, Proliferation Assay, Control