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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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Current recovery Q3,50 ms/Q1 (see text) for the different durations of the second pulse and 200 ms (protocol I, squares) or 10 s (protocol II, crosses) resting period.
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pone-0103874-g007: Current recovery Q3,50 ms/Q1 (see text) for the different durations of the second pulse and 200 ms (protocol I, squares) or 10 s (protocol II, crosses) resting period.

Mentions: To investigate current recovery in our data, we calculated for each cell the amount of Ca2+ influx during the first 50 ms of the third pulse (Q3,50 ms), and related it to the Ca2+ influx during the 50 ms prepulse (Q1). Mean recovery was then defined as the average of the ratios Q3,50 ms/Q1. We found that following a 50 ms prepulse and a 50 or 100 ms second pulse, the Ca2+ current did not inactivate much, and hence recovered substantially (mean recovery >75%; Fig. 7) in just 200 ms. In contrast, following longer second pulses the current did not recover much during 200 ms resting period (protocol I, squares in Fig. 7), but recovered almost fully in 10 s (protocol II; crosses in Fig. 7). For example, following the 50 ms prepulse and 800 ms second pulse, mean current recovery was 0.31±0.02 in protocol I and 0.87±0.01 in protocol II. This is in agreement with investigations of Ca2+ current recovery in mouse beta-cells [40].


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)

Current recovery Q3,50 ms/Q1 (see text) for the different durations of the second pulse and 200 ms (protocol I, squares) or 10 s (protocol II, crosses) resting period.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103874-g007: Current recovery Q3,50 ms/Q1 (see text) for the different durations of the second pulse and 200 ms (protocol I, squares) or 10 s (protocol II, crosses) resting period.
Mentions: To investigate current recovery in our data, we calculated for each cell the amount of Ca2+ influx during the first 50 ms of the third pulse (Q3,50 ms), and related it to the Ca2+ influx during the 50 ms prepulse (Q1). Mean recovery was then defined as the average of the ratios Q3,50 ms/Q1. We found that following a 50 ms prepulse and a 50 or 100 ms second pulse, the Ca2+ current did not inactivate much, and hence recovered substantially (mean recovery >75%; Fig. 7) in just 200 ms. In contrast, following longer second pulses the current did not recover much during 200 ms resting period (protocol I, squares in Fig. 7), but recovered almost fully in 10 s (protocol II; crosses in Fig. 7). For example, following the 50 ms prepulse and 800 ms second pulse, mean current recovery was 0.31±0.02 in protocol I and 0.87±0.01 in protocol II. This is in agreement with investigations of Ca2+ current recovery in mouse beta-cells [40].

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