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

GLP-1-stimulated ERK activation in MIN6 cells is mediated via local Ca²⁺ signalling.

<p>MIN6 cells incubated in KRB plus 1 mM glucose were loaded with 100 µM EGTA-AM or BAPTA-AM at room temperature prior to treatment with 10 nM GLP-1 plus 16.7 mM glucose for the times indicated. a) Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK2 (ERK2) 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; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. b) MIN6 cells were treated as in a) but in addition, loaded with 2 µM fura-2-AM and [Ca²⁺]_i levels measured by epifluorescence microscopy. i) The mean increase in [Ca²⁺]_i represented as area under the curve (A.U.C.) during ii) 10 min or iii) 30 min stimulation, mean +S.E.M. (n>30). Statistical comparisons were by one-way ANOVA with Dunnett's range test compared to GLP-1 plus glucose in the absence of chelator. ***, <i>P</i><0.001.</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

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

GLP-1-stimulated ERK activation requires L-type VGCC activation.

<p>a) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were then incubated in 16.7 mM glucose in the absence or presence of 10 nM GLP-1. 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 above densitometric analysis of the results showing mean +S.E.M. (n = 5). All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to glucose alone at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. b) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were stimulated with 10 nM GLP-1 plus 16.7 mM glucose in the presence or absence of 10 µM nifedipine for the times indicated. 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). All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose at each time point; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. c) MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. Cells were stimulated with 10 nM GLP-1 plus 16.7 mM glucose in the presence or absence of 50 µM dilitiazem for the times indicated. Cell lysates were analysed by SDS-PAGE and Western blotting using anti-phospho-ERK1/2 or anti-ERK2 antibodies. A representative blot is shown above densitometric analysis of the results ± S.E.M. All statistical comparisons were by one-way ANOVA with Bonferroni's multiple comparison test compared to glucose plus GLP1; *, <i>P</i><0.05; **, <i>P</i><0.01; (n = 3). d) MIN-6 cells were loaded with fura-2-AM and [Ca²⁺]_i levels measured using epifluorescence microscopy. i) Representative traces from single cells incubated with 1 mM glucose (control), 16.7 mM glucose (glucose), 10 nM GLP-1 plus 16.7 mM glucose (GLP-1/glucose) or 10 nM GLP-1 plus 16.7 mM glucose in the presence of 10 µM nifedipine (GLP-1/glucose + nifedipine). ii) Area under the curve (A.U.C.) across the 30 min stimulation showing mean +S.E.M. (n>30). Statistical comparisons were by one-way ANOVA with Dunnett's range test compared to GLP-1 plus glucose; ***, <i>P</i><0.001. e) Membrane potential recordings from MIN6 cells recorded in the perforated-patch, current-clamp mode. i) The effect of addition of 10 nM GLP-1 plus 16.7 mM glucose on membrane potential. ii) A membrane potential recording after 5.5 min in the continuous presence of 10 nM GLP-1 and 16.7 mM glucose. iii) After 10 min in the presence of 10 nM GLP-1 and 16.7 mM glucose, the effect on excitability of bath application of nifedipine (10 µM) was recorded.</p

The sustained activation of L-type VGCCs is required for the sustained activation of ERK.

<p>MIN6 cells were preincubated for 1 h in KRB supplemented with 1 mM glucose. The cells were then incubated in KRB containing 10 nM GLP-1 and 16.7 mM glucose in the absence or presence of 10 µM nifedipine applied at either 0, 10 or 20 min post-GLP-1/glucose addition. Proteins were resolved by SDS-PAGE and Western blotted using anti-phospho-ERK1/2 (pERK) and anti-ERK2 (ERK2) antibodies. A representative blot is shown with densitometric analysis of the results below showing mean +S.E.M. (n = 3). Results were analysed using two-way ANOVA with Bonferroni's multiple comparison test compared to GLP-1 plus glucose; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p