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Membrane potential-dependent inactivation of voltage-gated ion channels in alpha-cells inhibits glucagon secretion from human islets.

Ramracheya R, Ward C, Shigeto M, Walker JN, Amisten S, Zhang Q, Johnson PR, Rorsman P, Braun M - Diabetes (2010)

Bottom Line: Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude.Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV.We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK. matthias.braun@drl.ox.ac.uk

ABSTRACT

Objective: To document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release.

Research design and methods: Glucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry.

Result: Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis.

Conclusions: Human alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

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Voltage-gated TTX-sensitive Na+-channels. Experiments were performed in the presence of TEA (10 mmol/l) in the extracellular solution and after replacing K+ with Cs+ in the pipette solution. A: Currents recorded under control conditions, after addition of 1 mmol/l Co2+ and after addition of TTX (0.1 μg/ml) in the continued presence of Co2+. B: Voltage dependence of Na+-currents. The responses recorded in the presence of Co2+ during depolarizations to −40, −30, −20, and −10 mV are shown. C: I–V relationship for Na+-currents (n = 5). D: Inactivation of Na+-current. A test pulse to +10 mV was preceded by 50-ms conditioning pulses to membrane potentials between −150 and 0 mV (−60 to −30 shown). Currents were recorded in the presence of Co2+. E: Inactivation curve. The response after a conditioning pulse to −150 mV was taken as unity (n = 6). A Boltzmann function fit to the mean data has been superimposed. F: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of TTX (0.1 μg/ml) at 1 or 20 mmol/l glucose as indicated. 100% = 12.2 ± 3.8 pg/islet/h (n = 15; 4 donors). *P < 0.05 versus 1 mmol/l glucose alone.
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Figure 4: Voltage-gated TTX-sensitive Na+-channels. Experiments were performed in the presence of TEA (10 mmol/l) in the extracellular solution and after replacing K+ with Cs+ in the pipette solution. A: Currents recorded under control conditions, after addition of 1 mmol/l Co2+ and after addition of TTX (0.1 μg/ml) in the continued presence of Co2+. B: Voltage dependence of Na+-currents. The responses recorded in the presence of Co2+ during depolarizations to −40, −30, −20, and −10 mV are shown. C: I–V relationship for Na+-currents (n = 5). D: Inactivation of Na+-current. A test pulse to +10 mV was preceded by 50-ms conditioning pulses to membrane potentials between −150 and 0 mV (−60 to −30 shown). Currents were recorded in the presence of Co2+. E: Inactivation curve. The response after a conditioning pulse to −150 mV was taken as unity (n = 6). A Boltzmann function fit to the mean data has been superimposed. F: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of TTX (0.1 μg/ml) at 1 or 20 mmol/l glucose as indicated. 100% = 12.2 ± 3.8 pg/islet/h (n = 15; 4 donors). *P < 0.05 versus 1 mmol/l glucose alone.

Mentions: Voltage-gated inward currents were studied using Cs2+-containing pipette solution and TEA-containing bath solution to block K+-currents. Figure 4A shows membrane currents elicited by 5-ms depolarizations from −70 to 0 mV. Under control conditions, the response consisted of an initial transient component followed by a sustained current. The sustained current was inhibited by the broad-spectrum Ca2+-channel blocker Co2+ (1 mmol/l). In the presence of Co2+, a rapidly activating and inactivating current was observed that was inhibited by the Na+-channel blocker TTX.


Membrane potential-dependent inactivation of voltage-gated ion channels in alpha-cells inhibits glucagon secretion from human islets.

Ramracheya R, Ward C, Shigeto M, Walker JN, Amisten S, Zhang Q, Johnson PR, Rorsman P, Braun M - Diabetes (2010)

Voltage-gated TTX-sensitive Na+-channels. Experiments were performed in the presence of TEA (10 mmol/l) in the extracellular solution and after replacing K+ with Cs+ in the pipette solution. A: Currents recorded under control conditions, after addition of 1 mmol/l Co2+ and after addition of TTX (0.1 μg/ml) in the continued presence of Co2+. B: Voltage dependence of Na+-currents. The responses recorded in the presence of Co2+ during depolarizations to −40, −30, −20, and −10 mV are shown. C: I–V relationship for Na+-currents (n = 5). D: Inactivation of Na+-current. A test pulse to +10 mV was preceded by 50-ms conditioning pulses to membrane potentials between −150 and 0 mV (−60 to −30 shown). Currents were recorded in the presence of Co2+. E: Inactivation curve. The response after a conditioning pulse to −150 mV was taken as unity (n = 6). A Boltzmann function fit to the mean data has been superimposed. F: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of TTX (0.1 μg/ml) at 1 or 20 mmol/l glucose as indicated. 100% = 12.2 ± 3.8 pg/islet/h (n = 15; 4 donors). *P < 0.05 versus 1 mmol/l glucose alone.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927942&req=5

Figure 4: Voltage-gated TTX-sensitive Na+-channels. Experiments were performed in the presence of TEA (10 mmol/l) in the extracellular solution and after replacing K+ with Cs+ in the pipette solution. A: Currents recorded under control conditions, after addition of 1 mmol/l Co2+ and after addition of TTX (0.1 μg/ml) in the continued presence of Co2+. B: Voltage dependence of Na+-currents. The responses recorded in the presence of Co2+ during depolarizations to −40, −30, −20, and −10 mV are shown. C: I–V relationship for Na+-currents (n = 5). D: Inactivation of Na+-current. A test pulse to +10 mV was preceded by 50-ms conditioning pulses to membrane potentials between −150 and 0 mV (−60 to −30 shown). Currents were recorded in the presence of Co2+. E: Inactivation curve. The response after a conditioning pulse to −150 mV was taken as unity (n = 6). A Boltzmann function fit to the mean data has been superimposed. F: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of TTX (0.1 μg/ml) at 1 or 20 mmol/l glucose as indicated. 100% = 12.2 ± 3.8 pg/islet/h (n = 15; 4 donors). *P < 0.05 versus 1 mmol/l glucose alone.
Mentions: Voltage-gated inward currents were studied using Cs2+-containing pipette solution and TEA-containing bath solution to block K+-currents. Figure 4A shows membrane currents elicited by 5-ms depolarizations from −70 to 0 mV. Under control conditions, the response consisted of an initial transient component followed by a sustained current. The sustained current was inhibited by the broad-spectrum Ca2+-channel blocker Co2+ (1 mmol/l). In the presence of Co2+, a rapidly activating and inactivating current was observed that was inhibited by the Na+-channel blocker TTX.

Bottom Line: Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude.Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV.We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Diabetes Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK. matthias.braun@drl.ox.ac.uk

ABSTRACT

Objective: To document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release.

Research design and methods: Glucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry.

Result: Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis.

Conclusions: Human alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

Show MeSH
Related in: MedlinePlus