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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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Illustration of the protocol where a 50 ms prepulse is followed by depolarizations of varying length (50, 100, 200, 400, and 800 ms).The applied changes in membrane potential due to the depolarizations (V; top) evoke Ca2+-currents (ICa; middle) and increases in membrane capacitance (ΔCm; bottom). The panels show the data from a single cell in response to 3 (50 ms prepulse + 50, 200 or 800 ms depolarization) of the 5 double-pulses applied to the cell. We analyzed the data from the depolarizations of varying length following the prepulse.
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pone-0103874-g005: Illustration of the protocol where a 50 ms prepulse is followed by depolarizations of varying length (50, 100, 200, 400, and 800 ms).The applied changes in membrane potential due to the depolarizations (V; top) evoke Ca2+-currents (ICa; middle) and increases in membrane capacitance (ΔCm; bottom). The panels show the data from a single cell in response to 3 (50 ms prepulse + 50, 200 or 800 ms depolarization) of the 5 double-pulses applied to the cell. We analyzed the data from the depolarizations of varying length following the prepulse.

Mentions: The double-pulse data set analyzed above is a subset of a larger dataset where 50 ms prepulses followed by 100 ms resting periods at −70 mV and depolarizations of varying lengths (50, 100, 200, 400 and 800 ms), were applied to each cell (Fig. 5).


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)

Illustration of the protocol where a 50 ms prepulse is followed by depolarizations of varying length (50, 100, 200, 400, and 800 ms).The applied changes in membrane potential due to the depolarizations (V; top) evoke Ca2+-currents (ICa; middle) and increases in membrane capacitance (ΔCm; bottom). The panels show the data from a single cell in response to 3 (50 ms prepulse + 50, 200 or 800 ms depolarization) of the 5 double-pulses applied to the cell. We analyzed the data from the depolarizations of varying length following the prepulse.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103874-g005: Illustration of the protocol where a 50 ms prepulse is followed by depolarizations of varying length (50, 100, 200, 400, and 800 ms).The applied changes in membrane potential due to the depolarizations (V; top) evoke Ca2+-currents (ICa; middle) and increases in membrane capacitance (ΔCm; bottom). The panels show the data from a single cell in response to 3 (50 ms prepulse + 50, 200 or 800 ms depolarization) of the 5 double-pulses applied to the cell. We analyzed the data from the depolarizations of varying length following the prepulse.
Mentions: The double-pulse data set analyzed above is a subset of a larger dataset where 50 ms prepulses followed by 100 ms resting periods at −70 mV and depolarizations of varying lengths (50, 100, 200, 400 and 800 ms), were applied to each cell (Fig. 5).

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