35 research outputs found
cAMP dynamics in adult mouse ventricular Epac1-camps expressing cardiomyocytes upon treatment with cAMP elevating agents and PDE4 inhibition.
<p><b>(A)</b> Representative calcium traces in Fura2-AM loaded Epac1-camps transgenic cardiomyocytes under resting conditions (left) and upon electric field stimulation at 1 Hz (right). <b>(B)</b> Non-normalized FRET ratios do not differ between resting and paced Epac1-camps cardiomyocytes under basal and stimulated conditions (isoproterenol—ISO, 100 nM—plus 3-isobutyl-1-methylxathin—IBMX, 100 μM). <b>(C)</b> Representative FRET traces from Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or <b>(E)</b> with the direct AC activator forskolin (10 μM) leading to an increase of cAMP visualized as a decrease in the FRET ratio. Inhibition of PDE4 by rolipram (Roli, 10 μM) strongly enhances this effect, whereas the unselective PDE inhibitor IBMX (100 μM) has only little additional effect. <b>(D and F)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes. Cells were paced at 1 Hz. Values are means ± SEM; from n = 6 cells isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p
Ca<sup>2+</sup> transient amplitudes in ISO and forskolin treated cardiomyocytes.
<p>Cells were loaded with Fura2-AM, paced at 1 Hz and treated with 100 nM ISO or 10 μM forskolin with subsequent applications of PDE inhibitors rolipram 10 μM, 8-MMX 30 μM and IBMX 100 μM. Shown are baseline Ca<sup>2+</sup> amplitudes (black bars) and systolic Ca<sup>2+</sup> transient amplitudes (grey bars) measured in paced <b>(A)</b> and resting <b>(B)</b> cells. Means ± SEM, *—p<0.05; **—p<0.01; ***—p<0.001 with ANOVA (compared to control, second value after / as compared to previous stimulation) followed by the Gasser-Greenhouse correction. n = 7 for ISO and n = 9 for forskolin cells in A, and n = 4 and 5 for ISO and forskolin cells in B, respectively (all isolated from at least 3 mice for each condition). Effects of ISO and forskolin in A are significantly different (p = 0.03 by one-way ANOVA).</p
cAMP dynamics in adult mouse cardiomyocytes upon preincubation with IBMX and after low concentration of ISO.
<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes preincubated with 3-isobutyl-1-methylxanthin (IBMX, 100 μM) and then stimulated with non-saturating concentrations of the β-AR agonist isoproterenol (ISO, 1nM). The AC activator forskolin (10 μM) was used to reach the maximal FRET response. <b>(B)</b> Representative control traces (n = 4 and 8 for unpaced and paced, respectively) showing FRET response to IBMX response over the whole time-course involved in these experiments. Representative FRET responses (n = 7 each) to 1 nM ISO applied along (without IBMX prestimulation), followed by forskolin plus IBMX <b>(D)</b> Quantification of experiments from A and C shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with IBMX and ISO. Cells were paced at 1 Hz. Values are means ± SEM, n = 8 cells for each A graph and n = 7 cells for each C graph isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p
Subsarcolemmal cAMP dynamics in adult mouse ventricular cardiomyocytes transgenically expressing pmEpac1 biosensor.
<p><b>(A)</b> Representative FRET traces from pmEpac1 cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) and subsequently by the PDE4 inhibitor rolipram (Roli, 10 μM) followed by the unselective PDE inhibitor IBMX (100 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167974#pone.0167974.g001" target="_blank">Fig 1C and 1D</a>. <b>(C)</b> Representative FRET traces from pmEpac1 cardiomyocytes stimulated with ISO (100 nM) and subsequently by the PDE1 inhibitor 8-MMX (30 μM) followed by the unselective PDE inhibitor IBMX (100 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167974#pone.0167974.g003" target="_blank">Fig 3A and 3B</a>. <b>(B and D)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes treated with ISO, rolipram or 8-MMX. Cells were paced at 1 Hz. Values are means ± SEM; from n = 12 and n = 11 cells (unpaced and paced, respectively) isolated from 3 hearts per condition in B and n = 9 cell from 2 hearts each in D; n.s.—not significant by one-way ANOVA.</p
Schematic diagram highlighting Ca<sup>2+</sup> and cAMP changes observed in this study under different experimental conditions (basal, ISO and forskolin stimulated cardiomyocytes with and without pacing).
<p>Pacing leads to an increase in Ca<sup>2+</sup> levels which is further augmented by forskolin>ISO via PKA-dependent phosphorylation of Ca<sup>2+</sup> handling proteins. However, pacing has little effect on cAMP levels, apart from the case when it is combined with forskolin stimulation, together both lead to PDE1 activation. Increase of PDE1 activity affects presumably a discrete subcellular microdomain which constitutes a small percentage of the whole cellular AMP content and can be therefore revealed in the cytosol only by the use of a PDE1 inhibitor. Forskolin and ISO generate quantitatively comparable but differently shaped amounts of cAMP which may come from ISO-induced dissociation of PDE4D8 from the β<sub>1</sub>-adrenergic receptor. This mechanism regulates local second messenger pool at the receptor and allows more rapid increase of cAMP in the cytosol, as compared to forskolin stimulation.</p
Mutation of S and T residues in the third intracellular loop increases intracellular cAMP.
<p><b>A</b>) Schematic identifying potential serine and threonine residues in the third intracellular loop and C-terminus that could be targeted for regulation by phosphorylation. These residues were mutated to alanine to create 3 mutants: ST/A, S1-6A, and ST/A+S1-6A. <b>B</b>) GPR3-HA WT and mutants were transfected into HEK293 cells and harvested 24 hr later for cAMP EIA. (*) indicates a significant increase in cAMP level compared to “WT”. Significance was determined by Repeated Measures ANOVA followed by Dunnett’s Multiple Comparison Test (<i>p</i><0.01). Results are presented as mean ± S.E.M. from 6 separate experiments. <b>C)</b> 24 hr after transfection, cell surface proteins were biotinylated, precipitated, and surface expression of GPR3-HA was detected by Western blotting. 0.5 µg of total lysate was used to detect total GPR3 and β-actin expression. Blot is representative of 3 separate experiments: WT, lane 1; ST/A, lane 2; S1-6A, lane 3; ST/A+S1-6A, lane 4. <b>D–E</b>) Bands corresponding to surface GPR3 and total GPR3 expression were analyzed using densitometry. Densitometric values for total expression of GPR3 WT and mutants were normalized to β-actin (<b>D</b>). The densitometric value for surface GPR3 was divided by the densitometric value for total GPR3 expression and normalized to β-actin to compare the amount of GPR3 at the surface vs. total GPR3 (<b>E</b>). (*) indicates a significant decrease in surface expression of GPR3 mutants compared to “WT”. Significance was determined by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (<i>p</i><0.05). Results are presented as mean ± S.E.M from 3 separate experiments.</p
cAMP dynamics in adult mouse cardiomyocytes upon treatment with cAMP elevating agents and PDE1 inhibition.
<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or with the direct AC activator forskolin (10 μM) <b>(C)</b>. Subsequent application of the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX, 30 μM) enhances the cAMP stimulatory effect of ISO and forskolin. Stimulation with the unselective PDE inhibitor 3-isobutyl-1-methylxanthin (IBMX, 100 μM) leads to a further increase of cAMP. <b>(B and D)</b> Quantification of experiments shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with ISO. Forskolin stimulated cardiomyocytes show significant differences in PDE1 contribution to total PDE inhibition which is significantly higher in paced cardiomyocytes as compared to resting cells. Pretreatment of resting cardiomyocytes with calcium elevating reagents such as thapsigargin (100 nM) and calcium ionophore A23187 (10 μM) mimics the effect of field stimulation. Cells were paced at 1 Hz. Values are means ± SEM; n = 6–10 cells isolated from 3 hearts per condition; *—significant difference at p<0.05 by one-way ANOVA; n.s.- not significant.</p
Inhibition of endocytosis increases intracellular cAMP.
<p><b>A)</b> HEK293 cells were co-transfected with GPR3-HA and pcDNA
or with Dyn WT or Dyn K44A. Twenty-four hr after transfection, cells
were harvested for cAMP EIA. Bars with different letters are
significantly different. Significance was determined by One-way ANOVA
followed by Newman-Keuls multiple comparison test
(<i>p</i><0.01).Results are presented as mean ±
S.E.M from 4 separate experiments. <b>B–C</b>) HEK293 cells
were co-transfected with GPR3-RFP and CFP-Epac1(δDEP-CD)-YFP and
pcDNA, or Dyn WT or Dyn K44A. The YFP/CFP ratios were measured before
and after isoproterenol and IBMX treatment. <b>B)</b> Percent
change in the YFP/CFP ratio. <b>C</b>) FRET measurements were
converted to cAMP levels (µM) as described in the experimental
protocol. Bars with different letters are significantly different.
Significance was determined by One-way ANOVA followed by Newman-Keuls
Multiple Comparison Test (<i>p</i><0.01). Results were
obtained from 15 different GPR3-RFP expressing cells per group.</p
Overexpression of GRK2 and β-arrestin-2 decreases intracellular cAMP levels.
<p><b>A</b>) HEK293 cells were co-transfected with GPR3-HA and pcDNA or GPR3-HA and GRK2 and β-arrestin-2. Twenty-four hr after transfection, cells were harvested for cAMP EIA. Bars with different letters are significantly different. Significance was measured by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (<i>p</i><0.05). Results are presented as mean ± S.E.M from 3 separate experiments. <b>B–C</b>) HEK293 cells were co-transfected with GPR3-RFP, CFP-Epac1(δDEP-CD)-YFP, and pcDNA or GPR3-RFP, CFP-Epac1(δDEP-CD)-YFP, GRK2, and β-arrestin-2. The YFP/CFP ratios were measured before and after forskolin and IBMX treatment. <b>B)</b> Percent change in the YFP/CFP ratio. <b>C</b>) FRET measurements were converted to cAMP levels (µM) as described in the experimental protocol. (*) indicates a significant difference in the % change in YFP/CFP ratio or cAMP levels compared to “GPR3-RFP+pcDNA”. Significance was determined by Student’s <i>t</i> test (<i>p</i><0.05). Results were obtained from 13–18 different GPR3-RFP expressing cells per group.</p
Inhibition of endocytosis increases cell surface GPR3.
<p>(<b>A and B</b>) HEK293 cells were transfected with Dyn WT (<b>A</b>) or Dyn K44A (<b>B</b>) and 24 hr later, the cells were incubated with Alexa Fluor 488-labelled transferrin and imaged with a confocal microscope. Images are representative of 3 separate experiments. (<b>C–E</b>) Cells were co-transfected with GPR3-RFP and pcDNA (<b>C</b>), or Dyn WT (<b>D</b>) or Dyn K44A (<b>E</b>) and imaged using a confocal microscope 24 hr later. Bar = 10 µm. (<b>F</b>) One µg of HEK293 lysate was used to detect Dyn WT and Dyn K44A overexpression by Western blot. (<b>G</b>) HEK293 cells were co-transfected with GPR3-HA and Dyn WT or Dyn K44A and 24 hr after transfection cell surface proteins were biotinylated, precipitated, and surface expression of GPR3-HA was detected by Western blot analysis. 0.5 µg of total lysate was used to detect total GPR3 and GAPDH expression. Blot is representative of 3 separate experiments. (<b>H–I</b>) Bands corresponding to surface GPR3 and total GPR3 expression were analyzed using densitometry. <b>H</b>) Densitometric values for total GPR3 expression in cell lysates were normalized to GAPDH. <b>I</b>) The densitometric value for surface GPR3 was divided by the densitometric value for total GPR3 expression and normalized to GAPDH to compare the amount of GPR3 at the surface vs. total GPR3 expression. (*) indicates a significant increase in cell surface GPR3 compared to “GPR3+Dyn WT”. Significance was determined by Student’s <i>t</i> test (<i>p</i><0.05). Results are presented as mean ± S.E.M from 3 separate experiments.</p