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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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Related in: MedlinePlus

Mixed-effects analysis of the pulse-length data from the second pulse, following a 50 ms prepulse, in the control and EGTA groups.Capacitance data (ΔCm) plotted against Ca2+ influx measured as charge (Q) from individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Ctrl (panel 1–14); EGTA (panel 15–32).
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pone-0103874-g006: Mixed-effects analysis of the pulse-length data from the second pulse, following a 50 ms prepulse, in the control and EGTA groups.Capacitance data (ΔCm) plotted against Ca2+ influx measured as charge (Q) from individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Ctrl (panel 1–14); EGTA (panel 15–32).

Mentions: Linear mixed-effects modeling of ΔCm as a function of Q for the second pulse (Fig. 6) showed that the data could be fit with an intercept of zero, confirming that the prepulse depleted the small pool of ∼10 granules. The Ca2+ current sensitivity in the control group was 1.17±0.19 fF/pC (n = 13 cells), while it was significantly lower in the EGTA group (0.35±0.15 fF/pC, n = 18 cells; p<0.001 by t-test). Cell-to-cell variation was rather large as quantified by the standard deviation of 0.62 fF/pC for the random effect on Ca2+ current sensitivity. In summary, in the control group the Ca2+ current sensitivity was not reduced following a 50 ms prepulse (if anything, it tended to be slightly increased, 1.17±0.19 vs. 0.79±0.10 fF/pC, p = 0.088 by two-tailed t-test). Similarly, the application of a prepulse did not affect the Ca2+ current sensitivity in the EGTA group (0.35±0.15 vs. 0.36±0.09 fF/pC, p = 0.95). These results speak against pool depletion causing the capacitance pattern seen in Fig. 1A.


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 pulse-length data from the second pulse, following a 50 ms prepulse, in the control and EGTA groups.Capacitance data (ΔCm) plotted against Ca2+ influx measured as charge (Q) from individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Ctrl (panel 1–14); EGTA (panel 15–32).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103874-g006: Mixed-effects analysis of the pulse-length data from the second pulse, following a 50 ms prepulse, in the control and EGTA groups.Capacitance data (ΔCm) plotted against Ca2+ influx measured as charge (Q) from individual cells with single-cell fits indicated by solid lines, while group fits (fixed-effects) are given by the dashed lines. Ctrl (panel 1–14); EGTA (panel 15–32).
Mentions: Linear mixed-effects modeling of ΔCm as a function of Q for the second pulse (Fig. 6) showed that the data could be fit with an intercept of zero, confirming that the prepulse depleted the small pool of ∼10 granules. The Ca2+ current sensitivity in the control group was 1.17±0.19 fF/pC (n = 13 cells), while it was significantly lower in the EGTA group (0.35±0.15 fF/pC, n = 18 cells; p<0.001 by t-test). Cell-to-cell variation was rather large as quantified by the standard deviation of 0.62 fF/pC for the random effect on Ca2+ current sensitivity. In summary, in the control group the Ca2+ current sensitivity was not reduced following a 50 ms prepulse (if anything, it tended to be slightly increased, 1.17±0.19 vs. 0.79±0.10 fF/pC, p = 0.088 by two-tailed t-test). Similarly, the application of a prepulse did not affect the Ca2+ current sensitivity in the EGTA group (0.35±0.15 vs. 0.36±0.09 fF/pC, p = 0.95). These results speak against pool depletion causing the capacitance pattern seen in Fig. 1A.

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