Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes

Figures

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  Fig. 1. Natriuretic peptide effects on extracellular regulated kinase 1/2 (ERK1/2) phosphorylation and cGMP accumulation in GH3 cells. a Reverse transcription plus polymerase chain reaction (RT-PCR) analyses of GC-A (Npr1) and GC-B (Npr2) expression in GH3 cells. b, c Concentration-response studies of ERK1/2 phosphorylation (P-ERK1/2) or cGMP accumulation in response to (b) C-type natriuretric peptide (CNP) or (c) Atrial-type natriuretric peptide (ANP). In both instances, cells were stimulated for 15 min with the indicated concentrations of CNP prior to extraction of total proteins for Western blotting or total (intra- and extracellular) cGMP accumulation, as determined by cGMP enzyme immunoassay. Each autoradiograph is representative of at least three independent experiments; the scanning densitometry of ERK1/2 phosphorylation is pooled from at least three independent experiments. The cGMP data are expressed as the percentage of response to 10−7 M CNP or ANP for both ERK1/2 phosphorylation and cGMP accumulation. d RT-PCR analysis of Npr3 expression in GH3 cells and mouse gonadotrope αT3–1 and LβT2 cells (30 cycles). d’ GH3 cells were untreated (Basal) or treated with CNP (100 nM),…

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  Fig. 2. Time-course analysis of CNP and dibutryl-cGMP (db-cGMP) effects on ERK1/2 phosphorylation in GH3 cells. a, b GH3 cells were stimulated with (a) CNP (100 nM) or (b) db-cGMP (8-Br-cGMP; 1 mM) for up to 90 min, prior to extraction of total proteins and Western blotting for ERK1/2 phosphorylation. Each autoradiograph is representative of at least three independent experiments. c–e’ GH3 cells were treated for up to 90 min with 100 nM CNP prior to being fixed and stained for phospho-ERK1/2 (Alexa-488, green) or nuclear co-stained (DAPI 4,6-diamidino-2-phenylindole, blue). Immunofluorescence was visualised using confocal microscopy. Images shown are a representative field of vision from at least three independent experiments

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  Fig. 3. CNP stimulation of ERK1/2 phosphorylation is GC-B dependent. a RT-PCR analyses showing the optimisation of GC-B silencing by using short interfering RNA (siRNA). Total RNA was prepared from GH3 cells and reverse-transfected with the indicated concentrations of siRNA to GC-B or scrambled RNA control (20 nM) and harvested at 48 h post-transfection. b Western blotting to determine loss of GC-B protein in GC-B siRNA-treated GH3 cells (decreasing protein concentrations loaded per well). Total proteins were extracted at 48 h post-transfection and cell lysates were subjected to Western blot analyses for GC-B and then stripped and re-probed for β-Actin. c Western blotting analyses of GC-B siRNA-treated GH3 cells. GC-B siRNA or scrambled RNA-treated GH3 cells were stimulated with the indicated concentrations of CNP for 15 min. Cell lysates were subjected to Western blot analyses for phospho-ERK1/2 and then stripped and re-probed for total ERK1/2. d Specific silencing of GC-B1 and GC-B2 splice variant expression by using GC-B siRNA resulted in the loss of GC-B1 and GC-B2 (as determined by primers targeting the exon 20 to 22 region) but not GC-B1 and GC-B3 (as determined by primers…

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  Fig. 6. CNP stimulates αGSU promoter activity in a MEK-dependent manner. a GH3 cells were transiently transfected with 2.5 μg αGSU promoter and stimulated with the indicated concentrations of CNP or 1 mM db-cGMP. Cells were harvested after 8 h and protein extracts assayed for luciferase activity. Data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as fold increase over basal. b GH3 cells transiently transfected with αGSU promoter (as above) were pre-treated with the indicated concentration of U0126 for 30 min prior to stimulation with CNP (100 nM). Cells were harvested after 8 h and protein extracts assayed for luciferase activity. Data shown are means ± SEM of three independent experiments, each performed in triplicate and are expressed as fold increase over control response to CNP (*P < 0.1, ***P < 0.001, ****P < 0.0001; significantly different from control response, as determined by Bonferonni’s multiple comparisons test). c CNP enhances phosphorylation of ERK1/2 pathway proteins. GH3 cells were stimulated with CNP (100 nM) for up to 90 min, prior to extraction of total proteins and Western blotting for the indicated target…