Adrenaline stimulates glucagon secretion in pancreatic A-cells by increasing the Ca2+ current and the number of granules close to the L-type Ca2+ channels

Abstract

We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through omega-conotoxin-GVIA-sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via beta-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.

Figure 1

Spontaneous electrical activity in isolated rat pancreatic A-cells. (A) Overshooting action potentials generated by single A-cell in the absence of glucose. The dotted horizontal line indicates the zero-voltage level. (B) Voltage-gated inward currents evoked by membrane depolarizations from −90 to −15 mV or −70 to 0 mV as indicated. Presence of voltage-gated Na⁺ currents and N- and L-type Ca²⁺ channels revealed by inhibition by tetrodotoxin (0.3 μM) and ω-conotoxin-GVIA (1 μM) observed in the same cell and the inhibition by nifedipine (50 μM) observed in a different cell. Effects of ω-conotoxin-GVIA and nifedipine were tested in the presence of tetrodotoxin.

Figure 4

Relationship between Ca²⁺ entry and exocytosis. Ca²⁺ currents (middle) and changes in cell capacitance (bottom) evoked by 500-ms depolarizations from −70 to −10, +10, +30, and +50 mV applied at 30-s intervals before (A) and 2 min after (B) the addition of forskolin. (C) Increases in cell capacitance (ΔC_m) displayed against the integrated Ca²⁺ current (Q_ca) in the absence (filled symbols) and presence (open symbols) of forskolin. The different symbols refer to the responses evoked by 500-ms depolarizations from −70 to −10 (Δ, ▴), +10 (▿, ▾), +30 (□, ▪), and +50 mV (○, •). Data are presented as mean ± SEM of six separate experiments. The lines represent least-squares fit to the mean values. *P < 0.01 (vs. capacitance increase evoked by the depolarization to the same voltage under control conditions) and ^a P < 0.01 and ^b P < 0.02 (vs. integrated Ca²⁺ current elicited by the same depolarization under control conditions).

Figure 5

Voltage dependence of Ca²⁺ current amplitude, cytoplasmic Ca²⁺ transients, and exocytosis. (A). Ca²⁺ currents (I_Ca ,, second from top), increases in cell capacitance (C_m ,, second from bottom), and changes in the cytoplasmic Ca²⁺ concentration ([Ca^{2 +} ]_i ) elicited by voltage-clamp depolarizations going from −70 to +50, +30, +10, and −10 mV (top). Depolarizations were 500-ms long and were applied at ∼40-s intervals. (B) Depolarization-evoked integrated Ca²⁺ currents (Q_Ca ;, ▪), increases in cell capacitance (ΔC_m ;, ○), and cytoplasmic Ca²⁺ (Δ[Ca^{2 +} ]_i  ;, •) plotted against the voltage during the depolarizing commands.

Figure 7

Stimulation of exocytosis by photorelease of cAMP at fixed internal [Ca²⁺]_i. Effects of photoreleasing ∼30 μM cAMP from a caged precursor at 0.4 μM (A) and 0.17 μM (B) free intra-cellular Ca²⁺. The dotted line indicates the extrapolated rate of capacitance increase before the photorelease of cAMP in the presence of 0.4 μM [Ca²⁺]_i. The time at which photolysis of caged cAMP was effected is indicated by the vertical line.

Figure 3

β-adrenergic stimulation of Ca²⁺-dependent glucagon release involves activation of protein kinase A. (A) Simultaneous recordings of Ca²⁺ currents (second from top) and changes of [Ca²⁺]_i (second from bottom) and membrane capacitance (bottom) evoked by 500-ms depolarizations from −70 to 0 mV (top) before and 2 min after addition of the β-adrenergic agonist isoprenaline. (B) Histograms summarizing the effects of isoprenaline on the amplitude of the [Ca²⁺]_i-transient, the integrated Ca²⁺-current (Q_Ca), and the depolarization-evoked increase in cell capacitance (ΔC_m). (C) Ca²⁺ currents (middle) and changes in cell capacitance (bottom) evoked by 500-ms depolarizations from −70 to 0 mV before and 2 min after the addition of isoprenaline in the presence of Rp-8-Br-cAMPS (100 μM). In addition, the A-cell had been pretreated with PKA inhibitor for >20 min before the experiment. (D) Lack of effects of isoprenaline on the integrated Ca²⁺ current (Q_Ca) and the depolarization-evoked increase in cell capacitance (ΔC_m) in cells pretreated with Rp-8-Br-cAMPS. In A and C, the dotted lines above the current traces indicate the zero-current level. Data in B and D are mean ± SEM of indicated number (n) of experiments. *P < 0.05; **P < 0.01.

Figure 2

β-adrenergic stimulation of Ca²⁺-dependent exocytosis in A-cells. (A) Ca²⁺ currents (second from top), capacitance increases (second from bottom), and membrane conductance (bottom) evoked by 500-ms depolarizations from −70 to 0 mV before and 2 min after the addition of adrenaline. (B) Histograms summarizing the average effects of adrenaline on the integrated Ca²⁺ current (Q_Ca) and associated increases in cell capacitance (ΔC_m). (C) As in A but adrenaline was applied in the presence of the β-adrenergic antagonist propanolol. (D) As in B but data were obtained in the presence of propanolol. In A and C, the dotted lines above the current traces indicate the zero-current level. Data in B and D are mean ± SEM of indicated number (n) of experiments. *P < 0.025; **P < 0.01.

Figure 6

Increasing pulse duration results in saturation of exocytotic responses. (A) Ca²⁺ currents (middle) and changes in cell capacitance (bottom) evoked by 100-, 200-, 300-, and 500-ms depolarizations from −70 to 0 mV applied at 30-s intervals before (left) and 2 min after (right) the addition of forskolin. (B) Relationship between pulse duration and integrated Ca²⁺ current (Q_Ca ) in the absence (○) and presence (•) of 10 μM forskolin. The dotted lines are least-squares fits to the mean values of the currents elicited by depolarizations ≤300 ms. Note deviation from linearity at 500 ms. (C) Increases in cell capacitance (ΔC_m ) displayed against the integrated Ca²⁺ current (Q_ca ) in the absence (○) and presence (•) of forskolin evoked by depolarization between 50 and 500 ms. The data points have been approximated to the equation ΔC_m = ΔC_max * {1 − e^{−([Q − Qmin]/n)}} where ΔC_m is the observed capacitance increase, ΔC_max is the calculated maximum capacitance increase, Q is the associated charge entry, Q_min the calculated minimum integrated Ca²⁺ current required to evoke exocytosis and n is a slope factor. The function was approximated to each experiment and the values of ΔC_max, Q_min, and n calculated for each experiment. The fits were based on the responses to depolarizations ≤300 ms. The curves were drawn using the mean values of these parameters. These were (values obtained in the presence of forskolin given within the parentheses) 57 ± 15 fF (288 ± 64 fF), 0.7 ± 0.2 pC (1.0 ± 0.4 pC), and 7.5 ± 1.8 (3.8 ± 0.9) for ΔC_max, Q_min, and n, respectively. Data are mean ± SEM of five experiments. *P < 0.05; **P < 0.01 (vs. capacitance increase evoked by the same depolarization under control conditions).

Figure 8

Differential dependence of basal and stimulated exocytosis on Ca²⁺ influx through N- and L-type Ca²⁺ channels under basal conditions. (A) Ca²⁺ currents (middle) and changes in cell capacitance (bottom) evoked by membrane depolarizations from −90 to 0 mV (top) before and after addition of 1 μM ω-conotoxin-GVIA. (B) Same as A but the holding potential was −70 mV and nifedipine (5 μM) was applied. (C) Same as B but nifedipine was applied at a concentration of 50 μM. The holding potential was varied between −90 and −70 mV to prevent possible voltage-dependent inactivation of the ω-conotoxin–sensitive current. However, varying the holding potential does not influence the magnitude of the integrated Ca²⁺ current. In a series of five experiments, the integrated Ca²⁺ currents elicited by depolarizations to 0 mV using holding potentials of −90 and −70 mV amounted to 3.1 ± 0.5 and 2.9 ± 0.5 pC, respectively.

Figure 9

L-type Ca²⁺ channels mediate Ca²⁺ influx required for exocytosis in forskolin-stimulated A-cells. (A) Ca²⁺ currents (middle) and changes in cell capacitance (bottom) evoked by membrane depolarizations from −90 to 0 mV (top) before and after addition of 1 μM ω-conotoxin-GVIA. (B) Same as A, but the holding potential was −70 mV; forskolin (10 μM) was present throughout and the effects of 50 μM nifedipine were tested.

Figure 10

Model explaining the effects of the Ca²⁺ channel blockers. (A) Under basal conditions, most (65%) of the granules ready for release are localized in the vicinity of the N-type Ca²⁺ channels and exocytosis of these granules is blocked by addition of ω-conotoxin-GVIA. The shaded areas indicate the domains within which [Ca²⁺]_i rises to concentrations sufficient to trigger exocytosis. Note that there are more L- than N-type Ca²⁺ channels and the relative scarcity of granules within the domains of the L-type Ca²⁺ channels. (B) In the presence of forskolin, exocytosis is enhanced because (a) the Ca²⁺ current is increased and the domains around the L-type Ca²⁺ channels extend further into the A-cell; and (b) granules that were previously outside the domains have been “mobilized” and brought into closer proximity to the Ca²⁺ channels. By the combination of these effects, 80% of the releasable granules are now accessed by influx through the nifedipine-sensitive L-type Ca²⁺ channels. The extent of the domains under control condition is indicated by the dotted lines.