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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

No difference in Ca2+ current sensitivity between protocol I and protocol II.Capacitance increases are plotted vs. Ca2+ influx Q for the third 500-ms depolarizations following a 50 ms prepulse, a second pulse of either 200 ms (squares), 400 ms (circles) or 800 ms (triangles), and a resting period of either 200 ms (protocol I, black) or 10 s (protocol II, gray). The line indicates the fixed-effects fit from the linear mixed-effects model, which fitted the entire data set (both protocols).
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pone-0103874-g008: No difference in Ca2+ current sensitivity between protocol I and protocol II.Capacitance increases are plotted vs. Ca2+ influx Q for the third 500-ms depolarizations following a 50 ms prepulse, a second pulse of either 200 ms (squares), 400 ms (circles) or 800 ms (triangles), and a resting period of either 200 ms (protocol I, black) or 10 s (protocol II, gray). The line indicates the fixed-effects fit from the linear mixed-effects model, which fitted the entire data set (both protocols).

Mentions: Since our argumentation is based on substantial Ca2+ current inactivation, we limited our analysis to third pulses following a 50-ms prepulse and a second depolarization lasting ≥200 ms. These longer pulses would also favor the unmasking of any pool depletion, if it should occur. Mixed-effects modeling with ΔCm as a function of Q for third-pulse data with the second depolarization lasting ≥200 ms revealed no evidence of any difference between protocols I and II (p = 0.48 by likelihood ratio test, confirmed by the Akaike Information Criterion, between a model with intercept and slope depending on the protocol, and a simple model without protocol effect; Fig. 8). Moreover, the analysis gives results for the Ca2+ current sensitivity similar to the previous analyses of pulse-length data with or without prepulse (0.76±0.10 fF/pC). In summary, the exocytotic response recovers in parallel to Ca2+ currents, and the Ca2+ current sensitivity is unaffected by the two preceding pulses. These findings speak against pool depletion.


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)

No difference in Ca2+ current sensitivity between protocol I and protocol II.Capacitance increases are plotted vs. Ca2+ influx Q for the third 500-ms depolarizations following a 50 ms prepulse, a second pulse of either 200 ms (squares), 400 ms (circles) or 800 ms (triangles), and a resting period of either 200 ms (protocol I, black) or 10 s (protocol II, gray). The line indicates the fixed-effects fit from the linear mixed-effects model, which fitted the entire data set (both protocols).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103874-g008: No difference in Ca2+ current sensitivity between protocol I and protocol II.Capacitance increases are plotted vs. Ca2+ influx Q for the third 500-ms depolarizations following a 50 ms prepulse, a second pulse of either 200 ms (squares), 400 ms (circles) or 800 ms (triangles), and a resting period of either 200 ms (protocol I, black) or 10 s (protocol II, gray). The line indicates the fixed-effects fit from the linear mixed-effects model, which fitted the entire data set (both protocols).
Mentions: Since our argumentation is based on substantial Ca2+ current inactivation, we limited our analysis to third pulses following a 50-ms prepulse and a second depolarization lasting ≥200 ms. These longer pulses would also favor the unmasking of any pool depletion, if it should occur. Mixed-effects modeling with ΔCm as a function of Q for third-pulse data with the second depolarization lasting ≥200 ms revealed no evidence of any difference between protocols I and II (p = 0.48 by likelihood ratio test, confirmed by the Akaike Information Criterion, between a model with intercept and slope depending on the protocol, and a simple model without protocol effect; Fig. 8). Moreover, the analysis gives results for the Ca2+ current sensitivity similar to the previous analyses of pulse-length data with or without prepulse (0.76±0.10 fF/pC). In summary, the exocytotic response recovers in parallel to Ca2+ currents, and the Ca2+ current sensitivity is unaffected by the two preceding pulses. These findings speak against pool depletion.

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