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Calcium current inactivation rather than pool depletion explains reduced exocytotic rate with prolonged stimulation in insulin-secreting INS-1 832/13 cells.

Pedersen MG, Salunkhe VA, Svedin E, Edlund A, Eliasson L - PLoS ONE (2014)

Bottom Line: We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis.The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected.These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

View Article: PubMed Central - PubMed

Affiliation: Islet Cell Exocytosis, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden.

ABSTRACT
Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

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Analysis of the double-pulse data.The exocytotic response (ΔCm) is plotted against Ca2+-influx (charge; Q). Capacitance increases and Ca2+ influxes evoked by the first 50 ms-depolarization (Pulse 1) are shown as open symbols with fixed-effects fits indicated by dashed lines, while data evoked by the second 50-ms depolarization (Pulse 2) are plotted as filled symbols with their fixed-effects fits given by the solid lines. The colors and symbols indicate the groups (Black circles and lines: CTRL; Blue triangles and lines: EGTA). The graph contains data from n = 14 and n = 18 experiments from the control and EGTA group, respectively.
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pone-0103874-g004: Analysis of the double-pulse data.The exocytotic response (ΔCm) is plotted against Ca2+-influx (charge; Q). Capacitance increases and Ca2+ influxes evoked by the first 50 ms-depolarization (Pulse 1) are shown as open symbols with fixed-effects fits indicated by dashed lines, while data evoked by the second 50-ms depolarization (Pulse 2) are plotted as filled symbols with their fixed-effects fits given by the solid lines. The colors and symbols indicate the groups (Black circles and lines: CTRL; Blue triangles and lines: EGTA). The graph contains data from n = 14 and n = 18 experiments from the control and EGTA group, respectively.

Mentions: To circumvent this issue without neglecting the problem of Ca2+ current inactivation, we analyzed the capacitance increases ΔCm as a function of Ca2+ entry Q, derived under control conditions and in the presence of EGTA. A simple linear regression model with slope depending on both the group (CTRL/EGTA) and the pulse number revealed no statistically significant dependence on the pulse number (estimated slopes for the CTRL group: 1.18±0.28 vs. 0.42±0.31 fF/pC (p = 0.075); EGTA: 0.33±0.19 vs. 0.25±0.21 fF/pC (p = 0.79)). The data from the two groups were therefore further analyzed by a linear mixed-effects model with a slope depending on whether EGTA was present or not, but independent of the pulse number. In contrast, the intercept depended on the pulse number, but was not influenced by EGTA, and therefore common for the control and EGTA groups (Fig. 4). For the first pulse, the common intercept for the two groups was larger than zero (p<0.001) and estimated to be 6.94±1.68 fF. In contrast, for the second pulse the common intercept was estimated to be −0.40±1.59 fF, not statistically different from zero (p = 0.8), showing that the first pulse depleted the small pool. EGTA lowers the Ca2+ current sensitivity from 0.92±0.28 fF/pC in the control group to 0.26±0.19 fF/pC in the EGTA group (p<0.001). Together, these results confirm the presence of an IRP of ∼7 fF, which is unaffected by EGTA and depleted by the first depolarization. The exocytotic response to higher amounts of Ca2+ entry is reduced by EGTA and similar for the first and the second pulse, indicating that the first pulse does not deplete the pool responsible for later exocytosis.


Calcium current inactivation rather than pool depletion explains reduced exocytotic rate with prolonged stimulation in insulin-secreting INS-1 832/13 cells.

Pedersen MG, Salunkhe VA, Svedin E, Edlund A, Eliasson L - PLoS ONE (2014)

Analysis of the double-pulse data.The exocytotic response (ΔCm) is plotted against Ca2+-influx (charge; Q). Capacitance increases and Ca2+ influxes evoked by the first 50 ms-depolarization (Pulse 1) are shown as open symbols with fixed-effects fits indicated by dashed lines, while data evoked by the second 50-ms depolarization (Pulse 2) are plotted as filled symbols with their fixed-effects fits given by the solid lines. The colors and symbols indicate the groups (Black circles and lines: CTRL; Blue triangles and lines: EGTA). The graph contains data from n = 14 and n = 18 experiments from the control and EGTA group, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126658&req=5

pone-0103874-g004: Analysis of the double-pulse data.The exocytotic response (ΔCm) is plotted against Ca2+-influx (charge; Q). Capacitance increases and Ca2+ influxes evoked by the first 50 ms-depolarization (Pulse 1) are shown as open symbols with fixed-effects fits indicated by dashed lines, while data evoked by the second 50-ms depolarization (Pulse 2) are plotted as filled symbols with their fixed-effects fits given by the solid lines. The colors and symbols indicate the groups (Black circles and lines: CTRL; Blue triangles and lines: EGTA). The graph contains data from n = 14 and n = 18 experiments from the control and EGTA group, respectively.
Mentions: To circumvent this issue without neglecting the problem of Ca2+ current inactivation, we analyzed the capacitance increases ΔCm as a function of Ca2+ entry Q, derived under control conditions and in the presence of EGTA. A simple linear regression model with slope depending on both the group (CTRL/EGTA) and the pulse number revealed no statistically significant dependence on the pulse number (estimated slopes for the CTRL group: 1.18±0.28 vs. 0.42±0.31 fF/pC (p = 0.075); EGTA: 0.33±0.19 vs. 0.25±0.21 fF/pC (p = 0.79)). The data from the two groups were therefore further analyzed by a linear mixed-effects model with a slope depending on whether EGTA was present or not, but independent of the pulse number. In contrast, the intercept depended on the pulse number, but was not influenced by EGTA, and therefore common for the control and EGTA groups (Fig. 4). For the first pulse, the common intercept for the two groups was larger than zero (p<0.001) and estimated to be 6.94±1.68 fF. In contrast, for the second pulse the common intercept was estimated to be −0.40±1.59 fF, not statistically different from zero (p = 0.8), showing that the first pulse depleted the small pool. EGTA lowers the Ca2+ current sensitivity from 0.92±0.28 fF/pC in the control group to 0.26±0.19 fF/pC in the EGTA group (p<0.001). Together, these results confirm the presence of an IRP of ∼7 fF, which is unaffected by EGTA and depleted by the first depolarization. The exocytotic response to higher amounts of Ca2+ entry is reduced by EGTA and similar for the first and the second pulse, indicating that the first pulse does not deplete the pool responsible for later exocytosis.

Bottom Line: We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis.The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected.These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

View Article: PubMed Central - PubMed

Affiliation: Islet Cell Exocytosis, Lund University Diabetes Centre, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden.

ABSTRACT
Impairment in beta-cell exocytosis is associated with reduced insulin secretion and diabetes. Here we aimed to investigate the dynamics of Ca2+-dependent insulin exocytosis with respect to pool depletion and Ca2+-current inactivation. We studied exocytosis, measured as increase in membrane capacitance (ΔCm), as a function of calcium entry (Q) in insulin secreting INS-1 832/13 cells using patch clamp and mixed-effects statistical analysis. The observed linear relationship between ΔCm and Q suggests that Ca2+-channel inactivation rather than granule pool restrictions is responsible for the decline in exocytosis observed at longer depolarizations. INS-1 832/13 cells possess an immediately releasable pool (IRP) of ∼10 granules and most exocytosis of granules occurs from a large pool. The latter is attenuated by the calcium-buffer EGTA, while IRP is unaffected. These findings suggest that most insulin release occurs away from Ca2+-channels, and that pool depletion plays a minor role in the decline of exocytosis upon prolonged stimulation.

Show MeSH
Related in: MedlinePlus