Compound 2 potentiates GLP-1 9–36 amide-mediated cAMP generation by membranes from HEK-GLP-1R cells.

<p>Cell membranes prepared from HEK-GLP-1R cells were incubated with ligands as indicated for 5 min at 30°C in the presence of IBMX before determination of cAMP. A) Concentration-dependent cAMP generation in response to GLP-1 7-36 amide or to GLP-1 9–36 amide either alone or in combination with 3 µM compound 2. <b>B</b>) Responses to GLP-1 9–36 amide or compound 2 alone or the two in combination. The numerical sums of cAMP generation in response to GLP-1 9–36 amide and compound 2 are shown. Data are mean±/+s.e.m., n = 3. For * P<0.05 by Bonferroni's multiple range. For clarity only differences between ‘numerical’ and ‘co-addition’ conditions are shown.</p

Functional interaction of compound 2 and GLP-1 9–36 amide at the GLP-1R in HEK-GLP-1R cells.

<p>The pEC₅₀ values of GLP-1 9–36 amide-mediated cAMP generation in the presence of increasing concentrations of compound 2. The pEC₅₀ values have been determined from the data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047936#pone-0047936-g003" target="_blank">Figure 3</a>. Data are mean±s.e.m., n = 4, ** P<0.01 and *** P<0.001 versus 0 µM compound 2 by Dunnett's multiple range test following oneway ANOVA.</p

Time course of cAMP generation in response to GLP-1 9–36 amide, compound 2 or co-stimulation in HEK-GLP-1R cells.

<p>HEK-GLP-1R cells were either untreated (Basal; not visible) or treated for the indicated times with GLP-1 9–36 amide (1 µM), compound 2 (1 µM) or the two in combination (Co-addition) in the presence of IBMX. The final concentration of DMSO (vehicle) was 5% v/v in all cases. In addition to the measured levels of cAMP generation, the numerical sum of cAMP generation in response to GLP-1 9–36 amide and compound 2 alone are presented (Numerical). Data are mean±s.e.m., n = 3, ** P<0.01 and *** P<0.001 by Bonferroni's multiple range test following oneway ANOVA. For clarity, only differences between ‘numerical’ and ‘co-addition’ conditions are shown.</p

ERK signaling through the GLP-1R.

<p>HEK-GLP-1R cells (<b>A and B</b>) or INS-1E cells (<b>C and D</b>) were challenged for 5 min with either vehicle (1% v/v DMSO), GLP-1 9–36 amide (1 or 10 µM as indicated) or compound 2 alone or GLP-1 9–36 amide (1 µM) and compound 2 together as indicated. Cells were also challenged with GLP-1 7–36 amide (10 nM). Levels of phospho-ERK were then determined by Western blotting. The intensity of the bands representing phospho-ERK was determined using ImageJ and the mean data are shown in the panels below the immunoblot with basal (0) levels subtracted. Data are either representative of 3 experiments or mean+s.e.m., n = 3. *, P<0.05 and ** P<0.01 by Student's test versus the numerical sum of both GLP-1 9–36 amide (1 µM) and compound 2 at the concentration indicated when used alone.</p

Functional interaction between ligands on GLP-1R-mediated cAMP generation in HEK-GLP-1R cells.

<p>HEK-GLP-1R cells were pretreated (Pre-) for 10 min with 1 µM GLP-1 9–36 amide in the presence of IBMX before challenge for 15 min with the indicated concentrations of agonists. Where no pre-treatment is indicated, an equivalent volume of buffer (KHB) was added for 10 min in the presence of IBMX prior to ligand addition for 15 min. Levels of intracellular cAMP were then determined relative to the cellular protein content. The final concentration of DMSO (vehicle) for the 15 min treatment period was 5% v/v in all cases. Data are mean±s.e.m., n = 3.</p

Neutrophils express the class I PI3K catalytic subunits α,δ and γ.

<p>RNA was isolated from unstimulated neutrophils. RT-PCR was carried out using primers for each of the four class I PI3-kinase catalytic isoforms and β-actin as a control. n = 1 representative of three experiments. The identity of each of the bands was confirmed by excising the band and sequencing the DNA product.</p

A sustained global increase in [Ca²⁺]_i is insufficient for the prolonged activation of ERK.

<p>(a and b) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were incubated in 50 mM K⁺ (K50) in the presence or absence of 10 µM nifedipine for the times indicated (All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to K50 at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001). a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 and anti-ERK2 antibodies. A representative blot is shown above densitometric analysis of the results showing mean +S.E.M. (n = 3). b) In cells loaded with 2 µM fluo-4-AM, fluorescence (as an index of [Ca²⁺]_i) was measured using a NOVOstar platereader. (c and d) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were then treated with 10 µM ionomycin or 50 mM K⁺ (K50) for the times indicated. c) In cells loaded with 2 µM fluo-4-AM, fluorescence (as an index of [Ca²⁺]_i) was measured using a NOVOstar platereader. ***, <i>P</i><0.001 for K50 versus ionomycin at equivalent time points. d) After treatments, proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) or anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3).</p

The role of intracellular Ca²⁺ stores in GLP-1-stimulated ERK activation.

<p>a) MIN6 cells were either pre-incubated in KRB supplemented with 1 mM glucose in the absence (control) or presence of 100 µM ryanodine or 1 µM thapsigargin for 30 min prior to treatment with 10 nM GLP-1 plus 16.7 mM glucose for the times indicated. Where indicated, cells were also treated with 16.7 mM glucose alone. Proteins were separated by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK1/2 (ERK1/2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3). Data were analysed by two-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose at each time point. No significant differences were observed. b) MIN6 cells incubated in the absence of extracellular Ca²⁺ calcium were preincubated without or with 100 µM ryanodine for 30 min prior to the addition 10 mM caffeine. Changes in fluorescence as an index of [Ca²⁺]_i were determined in fluo-4-loaded cells using a NOVOstar platereader. c) MIN6 cells incubated in the absence of extracellular Ca²⁺ were pretreated without or with 1 µM thapsigargin for 30 min prior to the addition 100 µM carbachol (Carb.) . Changes in [Ca²⁺]_i were determined in fluo-4-loaded cells using a NOVOstar platereader ***, <i>P</i><0.001 by Student's t test.</p

Summary of the degree of inhibition obtained with the PI3 kinase inhibitors on CXCL-8 and GMCSF induced neutrophil migration.

<p>% inhibition</p><p>+++ ~100%</p><p>++ >50%</p><p>- Not significant</p><p>ND: Not done</p><p>Summary of the degree of inhibition obtained with the PI3 kinase inhibitors on CXCL-8 and GMCSF induced neutrophil migration.</p

L-type VGCC activation is sufficient to mediate sustained ERK activation in MIN6 cells via local Ca²⁺ signalling.

<p>MIN6 cells incubated in KRB plus 2 mM glucose were loaded with 100 µM of EGTA-AM or BAPTA-AM prior to treatment with 10 µM Bay-K 8644 at room temperature for the times indicated. a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) or anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 4). Results were analysed by two-way ANOVA with Bonferroni's multiple comparison test compared to Bay-K 8644 alone at each time point; ***, <i>P</i><0.001. b) MIN6 cells were treated as in (a) but in addition were loaded with 2 µM fura-2-AM and [Ca²⁺]_i levels measured by epifluorescence microscopy (n>30). A mean trace is shown. In addition, a representative trace obtained from MIN6 cells stimulated with 10 nM GLP-1 plus 16.7 mM glucose was included for comparison.</p