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

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.

FIG. 1.

Glucose dependence of glucagon secretion and effect of paracrine modulators. A–C: Secretion of glucagon (A), insulin (B), and somatostatin (C) measured at 1, 6, and 20 mmol/l glucose. Data are from 50 donors (glucagon) or 33 donors (insulin and somatostatin). *P < 0.05, **P < 0.01, ***P < 0.001 versus the previous lower glucose concentration. Glucagon secretion was significantly lower at 20 mmol/l compared with 1 mmol/l glucose (P < 0.05). D: Glucagon secretion measured at 1 and 6 mmol/l glucose under control conditions and in the presence of 100 nmol/l of CYN-154806 (CYN). 100% = 3.4 ± 0.4 pg glucagon/islet/h. E: As in D but in the absence and presence of 100 nmol/l insulin. 100% = 4.9 ± 0.9 pg glucagon/islet/h. F: As in D but in the absence and presence of 30 μmol/l ZnCl₂. 100% = 3.8 ± 0.5 pg glucagon/islet/h. D–F: *P < 0.05, **P < 0.01, ***P < 0.001 versus 1 mmol/l glucose (control) or as indicated by brackets.

FIG. 2.

Analysis of voltage-gated K⁺-currents. A: Family of voltage-activated K⁺-currents (lower) evoked by depolarizing pulses from −70 mV to membrane potentials between −40 and +80 mV. Inset shows inactivation of current during a 15-s depolarization from −70 to +20 mV. B: As in A but showing the initial part of the current responses during pulses to −40, −30, −20, and −10 mV on an expanded time base (sections highlighted in gray in A). C: I–V relationship for voltage-gated K⁺-current (n = 8). D: Example of a cell showing a clear shoulder on the I–V at voltages between +30 and +50 mV. Data are shown for the peak current (squares), the sustained current measured at the end of the 500-ms pulse (circles), and the difference (triangles).

FIG. 3.

Pharmacological characterization of voltage-gated K⁺-currents. A: Current responses recorded during depolarizations to +20 mV under control conditions, after addition of 10 mmol/l TEA (gray trace) and after addition of 5 mmol/l 4-aminopyridine in the continued presence of TEA (n = 4). B: As in A but pulse went to zero and currents were recorded in the absence and presence of stromatoxin (100 nmol/l, n = 4). C: As in A but pulse went to −10 mV and currents were recorded in the presence of 10 mmol/l TEA before and after application of heteropodatoxin-2 (0.5 μmol/l). D: Steady-state inactivation of the A-current analyzed by a two-pulse protocol consisting of a 200-ms conditioning pulse to membrane potentials between −90 and −20 mV followed by a 100-ms test pulse to +30 mV after an interval of 10 ms. Experiments were performed using the perforated-patch technique in the presence of TEA. E: Steady-state inactivation of the delayed-rectifying K⁺-current was measured by applying 15-s conditioning pulses to membrane potentials between −60 and −20 mV followed by a 500-ms test pulse to +20 mV (interval 10 ms). F: Voltage dependence of inactivation of A-current (closed circles) and delayed-rectifier current (open circles). The responses after conditioning pulses to −90 and −60 mV, respectively, were taken as unity, and data are presented as a fraction of the maximal current displayed against the voltage during the conditioning pulse. A Boltzmann function has been fit to the data points (n = 5–7). G: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of 0.5 μmol/l heteropodatoxin-2 at 1 or 6 mmol/l glucose. *P < 0.01 versus 1 mmol/l glucose alone. 100% = 6.5 ± 0.8 pg/islet/h (n = 12; 4 donors). H: Effects of 100 nmol/l stromatoxin on glucagon secretion at 1 or 20 mmol/l glucose. *P < 0.05 versus 1 mmol/l glucose. 100% = 7.5 ± 1.5 pg/islet/h (n = 9; 3 donors).

FIG. 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.

FIG. 5.

Voltage-gated Ca²⁺-currents. Experiments were performed with TEA-containing extracellular and Cs⁺-containing pipette solution. A: Family of voltage-gated Ca²⁺-currents recorded in the presence of TTX during 100-ms depolarizations to between −60 and 0 mV as indicated. B: Current voltage relationship of whole-cell Ca²⁺-currents (n = 14). C: Ca²⁺-current recorded under control conditions and after addition of 10 μmol/l isradipine and ω-conotoxin (100 nmol/l) and ω-agatoxin (200 nmol/l) in the continued presence of isradipine as indicated. D: Ca²⁺-currents elicited by voltage ramps (speed, 3 V/s) under control conditions and after addition of isradipine and ω-agatoxin in the continued presence of isradipine (n = 7, 7, 4 under control conditions, in the presence of isradipine, and after addition of ω-agatoxin, respectively). E: Isradipine- (top) and ω-agatoxin-sensitive components (lower) from D. F: Ca²⁺-currents elicited by voltage ramps in the presence of isradipine alone (10 μmol/l) and after addition of NNC 55-0396 (3 μmol/l) in the continued presence of isradipine. The difference current (T-type; gray) is also shown. G: Inactivation of the T-type Ca²⁺-current. A test pulse to −30 mV was preceded by 500-ms conditioning pulses to membrane potentials between −90 and −50 mV (in the presence of 10 μmol/l isradipine). H: Voltage-dependent inactivation of T-type Ca²⁺-current. The current elicited after a conditioning pulse to −100 mV was taken as unity. A Boltzmann fit has been superimposed on the data points (n = 6, experiments performed in the presence of isradipine).

FIG. 6.

Effects of Ca²⁺-channel antagonists on glucagon secretion. A: Glucagon secretion measured in the absence (open bars) and presence (filled bars) of 10 μmol/l isradipine. *P < 0.01 versus 1 mmol/l glucose alone, †P < 0.05 versus 1 mmol/l glucose and 10 μmol/l isradipine. 100% = 10.5 ± 0.6 pg/islet/h (n = 9; 3 donors). B: Same as in A but effects of 200 nmol/l ω-agatoxin were tested. *P < 0.01 versus 1 mmol/l glucose alone, 100% = 21.1 ± 3.7 pg/islet/h (n = 9; 3 donors).

FIG. 7.

Electrical activity and [Ca²⁺]_i oscillations. A: Membrane potential recording from an α-cell exposed to 1 mmol/l glucose. Note prominent after-hyperpolarizations after each action potential (arrows). B: Effect of tolbutamide on α-cell membrane potential in two representative cells. Note reduction of peak voltage. C: Spontaneous [Ca²⁺]_i oscillations in an α-cell within an intact islet exposed to 1 mmol/l glucose before and during addition of 5 μmol/l adrenaline. D: As in C but testing the effects of ω-agatoxin (200 nmol/l), isradipine (10 μmol/l), Bay K8644 (10 μmol/l), and K⁺ (70 mmol/l) at 1 mmol/l glucose. The inset shows the segment of the recording highlighted in gray on an expanded timebase. E: Histogram summarizing the average amplitude of the [Ca²⁺]_i oscillations under the indicated experimental conditions (13 cells in four islets obtained from two donors; *P < 0.01, **P < 0.001 vs. 1 mmol/l glucose or as indicated by brackets). F: As in D but ω-agatoxin was not applied. G: Histogram summarizing results obtained as described in F from 14 cells in four islets from two donors (**P < 0.001 vs. 1 mmol/l glucose).

FIG. 8.

Capacitance measurements of exocytosis. A: Increase in membrane capacitance evoked by 20–500-ms depolarizations from −70 to 0 mV. The circles above the capacitance traces indicate the percentage of responding cells (black part, n = 23). B: Relationship between pulse duration and exocytotic response (ΔC_m; n = 23). C: Change in cell capacitance (ΔC_m) evoked by 500-ms depolarizations from −70 mV to membrane potentials between −40 and +40 mV (n = 8). D: Change in cell capacitance evoked by 500-ms depolarization from −70 to 0 mV under control conditions and after addition of ω-agatoxin (200 nmol/l).