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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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Mixed-effects analysis of the train protocol.Cumulative capacitance data is plotted against cumulative Ca2+ influx measured as charge, for individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Model fit to all cumulative data from all 10 pulses is given in gray, while the fit to the first three pulses is given in black.
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pone-0103874-g009: Mixed-effects analysis of the train protocol.Cumulative capacitance data is plotted against cumulative Ca2+ influx measured as charge, for individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Model fit to all cumulative data from all 10 pulses is given in gray, while the fit to the first three pulses is given in black.

Mentions: Interestingly, and supporting our conclusion, repeated 500-ms pulses did not lead to the exhaustion of the increases in membrane capacitance in INS-1 832/13 cells (Fig. 9) [12]. However, we did find evidence of an exhaustible pool since our analysis of the first three pulses revealed a non-zero intercept ∼30 fF, which likely corresponds to the IRP found in the pulse-length and double-pulse protocols. It is not clear why the cells used for the train protocol have larger IRP, but the Ca2+ current sensitivity was also increased, suggesting that these cells were generally highly responsive. Note also that there was a large uncertainty in the estimate of the size of the IRP (standard error 8.6 fF) and a large cell-to-cell variation (the standard deviation of the random effect was 17.9 fF), indicating that the train protocol is not appropriate for determining the size of the IRP.


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)

Mixed-effects analysis of the train protocol.Cumulative capacitance data is plotted against cumulative Ca2+ influx measured as charge, for individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Model fit to all cumulative data from all 10 pulses is given in gray, while the fit to the first three pulses is given in black.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103874-g009: Mixed-effects analysis of the train protocol.Cumulative capacitance data is plotted against cumulative Ca2+ influx measured as charge, for individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Model fit to all cumulative data from all 10 pulses is given in gray, while the fit to the first three pulses is given in black.
Mentions: Interestingly, and supporting our conclusion, repeated 500-ms pulses did not lead to the exhaustion of the increases in membrane capacitance in INS-1 832/13 cells (Fig. 9) [12]. However, we did find evidence of an exhaustible pool since our analysis of the first three pulses revealed a non-zero intercept ∼30 fF, which likely corresponds to the IRP found in the pulse-length and double-pulse protocols. It is not clear why the cells used for the train protocol have larger IRP, but the Ca2+ current sensitivity was also increased, suggesting that these cells were generally highly responsive. Note also that there was a large uncertainty in the estimate of the size of the IRP (standard error 8.6 fF) and a large cell-to-cell variation (the standard deviation of the random effect was 17.9 fF), indicating that the train protocol is not appropriate for determining the size of the IRP.

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