Glucose and pharmacological modulators of ATP-sensitive K+ channels control [Ca2+]c by different mechanisms in isolated mouse alpha-cells

Abstract

Objective: We studied how glucose and ATP-sensitive K(+) (K(ATP)) channel modulators affect alpha-cell [Ca(2+)](c).

Research design and methods: GYY mice (expressing enhanced yellow fluorescent protein in alpha-cells) and NMRI mice were used. [Ca(2+)](c), the K(ATP) current (I(KATP), perforated mode) and cell metabolism [NAD(P)H fluorescence] were monitored in single alpha-cells and, for comparison, in single beta-cells.

Results: In 0.5 mmol/l glucose, [Ca(2+)](c) oscillated in some alpha-cells and was basal in the others. Increasing glucose to 15 mmol/l decreased [Ca(2+)](c) by approximately 30% in oscillating cells and was ineffective in the others. alpha-Cell I(KATP) was inhibited by tolbutamide and activated by diazoxide or the mitochondrial poison azide, as in beta-cells. Tolbutamide increased alpha-cell [Ca(2+)](c), whereas diazoxide and azide abolished [Ca(2+)](c) oscillations. Increasing glucose from 0.5 to 15 mmol/l did not change I(KATP) and NAD(P)H fluorescence in alpha-cells in contrast to beta-cells. The use of nimodipine showed that L-type Ca(2+) channels are the main conduits for Ca(2+) influx in alpha-cells. gamma-Aminobutyric acid and zinc did not decrease alpha-cell [Ca(2+)](c), and insulin, although lowering [Ca(2+)](c) very modestly, did not affect glucagon secretion.

Conclusions: alpha-Cells display similarities with beta-cells: K(ATP) channels control Ca(2+) influx mainly through L-type Ca(2+) channels. However, alpha-cells have distinct features from beta-cells: Most K(ATP) channels are already closed at low glucose, glucose does not affect cell metabolism and I(KATP), and it slightly decreases [Ca(2+)](c). Hence, glucose and K(ATP) channel modulators exert distinct effects on alpha-cell [Ca(2+)](c). The direct small glucose-induced drop in alpha-cell [Ca(2+)](c) contributes likely only partly to the strong glucose-induced inhibition of glucagon secretion in islets.

FIG. 1.

Glucose slightly decreases oscillating [Ca²⁺]_c in α-cells isolated from GYY (A–C) and NMRI mice (D, adrenaline-responsive cells). A–D: The glucose (G) concentration was changed between 0.5 and 15 mmol/l, and adrenaline (Adr, 10 μmol/l) was added as indicated. C and D: The perifusion medium was supplemented with a 2.5-mmol/l mixture of amino acids (mix AA). Data are means ± SE of results obtained in 21 (A), 35 (B), and 54 (C) isolated GYY α-cells and in 46 isolated NMRI α-cells (D).

FIG. 2.

Spontaneous [Ca²⁺]_c oscillations in isolated mouse α-cells are highly variable and result from synchronous and intermittent Ca²⁺-dependent spiking electrical activity. α-Cells were perifused with a medium containing 0.5 mmol/l glucose (G0.5) and a 2.5-mmol/l mixture of amino acids (mix AA). A: Examples of [Ca²⁺]_c oscillations in three isolated α-cells. B and C: The membrane potential (MP) was recorded in single α-cells in the perforated mode of the patch-clamp technique, and in B, it was simultaneously monitored with [Ca²⁺]_c. Note the overshoots in the spikes, a typical feature of α-cells (inset in B). In C, α-cells were first perifused with a medium containing 1.5 mmol/l Ca²⁺ (Ca1.5) and then, when indicated, with a Ca²⁺-free medium supplemented with 200 μmol/l EGTA (Ca0). Traces are representative of results obtained in 910 (A), 8 (B), and 3 (C) α-cells from GYY mice.

FIG. 3.

Effects of drugs on [Ca²⁺]_c in α-cells. Closure of K_ATP channels by tolbutamide induced [Ca²⁺]_c oscillations, whereas opening of K_ATP channels with diazoxide and blockade of cell metabolism with sodium azide lowered [Ca²⁺]_c to basal levels in isolated α-cells from GYY mice. The perifusion medium contained 0.5 (A--C and E) or 15 mmol/l (C and D) glucose (G) and a 2.5-mmol/l mixture of amino acids (mix AA) (C, top trace, and E). A: Sequential addition of 10 μmol/l tolbutamide (Tb) and 100 μmol/l diazoxide (Dz). B: Sequential addition of 100 μmol/l diazoxide (Dz) and 100 μmol/l tolbutamide (Tb). C: The diazoxide concentration (Dz) was increased stepwise, and 100 μmol/l tolbutamide (Tb) was applied as indicated. D: Arginine (Arg; 10 mmol/l) and 100 μmol/l diazoxide (Dz) were applied when indicated. E. Sodium azide (2 mmol/l), 500 μmol/l tolbutamide (Tb), and 10 μmol/l adrenaline (Adr) were applied as indicated. Traces are representative of results obtained in 9 (A), 8 (B), 8 (G15; C), 46 (G0.5 + mix AA; C), 12 (D), and 19 (E) α-cells from GYY mice.

FIG. 4.

α-Cells possess a I_KATP with similar characteristics to those of β-cells, except for its insensitivity to glucose. I_KATP was monitored by pulses of ±20 mV from a holding potential of −80 mV using the perforated mode of the patch-clamp technique. The current density was obtained by dividing the amplitude of the current by the membrane capacitance of the cell. I_KATP was recorded in β-cells (A) and α-cells (B) from GYY mice infected with AdRIPBgliDsRed, in noninfected β-cells from RIPYY mice (C), and in noninfected α-cells from GYY mice (D and E). The glucose (G) concentration was 15 mmol/l throughout (A-B and E), or changed between 0.5 and 15 mmol/l as indicated (C and D). Sodium azide (1 mmol/l in A and B or 2 mmol/l in E), 250 μmol/l diazoxide (Dz; A, B, D, and E), and 250 μmol/l tolbutamide (Tb; A and B) were applied when indicated. Each trace is representative of five experiments.

FIG. 5.

Glucose increases NAD(P)H fluorescence in isolated β-cells but not in isolated α-cells. NAD(P)H fluorescence is expressed as a percentage of the maximum signal recorded in the presence of azide. The glucose (G) concentration was changed between 0.5 and 15 mmol/l, and 5 mmol/l sodium azide was added when indicated. For all experiments, NAD(P)H fluorescence was first monitored. Thereafter, islet cells were loaded with fura-2/AM for 20 min on the stage of the microscope and then challenged with 10 μmol/l adrenaline. Cells that responded to adrenaline were considered as α-cells. A: α-Cells from GYY mice were identified by their EYFP fluorescence before NAD(P)H fluorescence measurement, and their [Ca²⁺]_c responsiveness to adrenaline was verified thereafter. The EYFP-negative cells were not responsive to adrenaline, and most of them were presumably β-cells. B: Isolated cells from NMRI mouse islets were monitored for NAD(P)H fluorescence, and their [Ca²⁺]_c responsiveness to adrenaline was verified thereafter. Adrenaline-responsive and -nonresponsive cells were considered as α- and β-cells, respectively. Data are means ± SE of results obtained in 30 α-cells and 14 EYFP-negative cells from GYY mice (A) and in 18 adrenaline-nonresponsive cells and 33 adrenaline-responsive cells from NMRI mice (B).

FIG. 6.

Glucose decreases [Ca²⁺]_c in α-cells independently from an action on K_ATP and VDCCs. The glucose (G) concentration was changed between 0.5 and 15 mmol/l, and 10 μmol/l adrenaline (Adr) was added as indicated. A: Tolbutamide (Tb; 500 μmol/l) was applied as indicated. B: The KCl concentration (K) in the perifusion medium was changed from 4.8 to 30 mmol/l as indicated. Data are means ± SE of results obtained in 26 (A) and 19 (B) α-cells from GYY mice.

FIG. 7.

GABA and zinc did not decrease α-cell [Ca²⁺]_c, and insulin, although lowering [Ca²⁺]_c very modestly, did not affect glucagon secretion. The medium contained a 2.5-mmol/l amino acid mixture (mix AA) and 0.5 mmol/l glucose (G). GABA (100 μmol/l), 3 or 30 μmol/l ZnCl₂, 100 nmol/l insulin, and 10 μmol/l adrenaline (Adr) were added when indicated. Data are means ± SE of results obtained in 31 (A), 7 (3 μmol/l ZnCl₂; B), 43 (30 μmol/l ZnCl₂; B), and 31 (C) α-cells from GYY mice and from four glucagon secretion experiments with 200 islets of GYY mice per chamber (D).

FIG. 8.

Spontaneous [Ca²⁺]_c oscillations and the rise in [Ca²⁺]_c elicited by tolbutamide or arginine result from Ca²⁺ influx through L-type channels in α-cells. The glucose concentration was 0.5 mmol/l throughout. Nimodipine (Nimo; 1 μmol/l), 10 μmol/l adrenaline (Adr; A-C), 100 μmol/l tolbutamide (Tb; B), and 10 mmol/l arginine (Arg; C) were added when indicated. Data are means ± SE of results obtained in 10 (A), 4 (B), and 7 (C) α-cells from GYY mice.