Glucagon activates Ca2+ and Cl- channels in rat hepatocytes

Abstract

Glucagon is one of the major hormonal regulators of glucose metabolism, counteracting the hepatic effects of insulin when the concentration of glucose in the bloodstream falls below a certain level. Glucagon also regulates bile flow, hepatocellular volume and membrane potential of hepatocytes. It is clear that changes in cell volume and membrane potential cannot occur without significant ion fluxes across the plasma membrane. The effects of glucagon on membrane currents in hepatocytes, however, are not well understood. Here we show, by patch-clamping of rat hepatocytes, that glucagon activates two types of currents: a small inwardly rectifying Ca2+ current with characteristics similar to those of the store-operated Ca2+ current and a larger outwardly rectifying Cl- current similar to that activated by cell swelling. We show that the mechanism of glucagon action on membrane conductance involves phospholipase C and adenylyl cyclase. Contribution of the adenylyl cyclase-dependent pathway to activation of the currents depended on Epac (exchange protein directly activated by cAMP), but not on protein kinase A. The activation of Ca2+ and Cl- channels is likely to play a key role in the mechanisms by which glucagon regulates hepatocyte metabolism and volume.

Figure 1. Effect of glucagon on membrane conductance in rat hepatocytes

A and B show the time course of the development of the inward and outward current in response to glucagon (10 nm) in perforated-patch (A, n = 5) and whole-cell experiments (B, n = 15). Current amplitudes were taken from the responses to the voltage ramps at −118 and +82 mV for the inward and the outward current, respectively, normalized to the cell capacitance and plotted against time. Application of glucagon is indicated by a horizontal bar. C, examples of the I–V plots obtained in response to voltage ramps between −138 and +102 mV before application of glucagon (trace 1, see A), at the beginning of the glucagon response (trace 2), and when the currents are fully developed (trace 3). D, lack of effect of the glucagon receptor antagonist des-His¹[Glu⁹]-glucagon amide (50 nm) on membrane conductance (n = 8). E, inhibition of the glucagon response (50 nm) by des-His¹[Glu⁹]-glucagon amide (50 nm; •; representative cell, n = 5). For comparison, a response to glucagon (50 nm) is shown in a representative cell from the same preparation (○).

Figure 2. Effects of ion substitution on glucagon-activated conductance

A, effect of Cl⁻ replacement in the external solution with an equimolar amount of glutamate on the outward current shown in a representative cell (n = 5). B, effect of replacement of Na⁺ in the external solution with NMDG⁺ on inward current shown in a representative cell (n = 4). NMDG⁺ was perfused both before and after development of the glucagon response.

Figure 3. Effects of removal of extracellular Ca²⁺ and addition of La³⁺ on the currents activated by glucagon

A and B show the effect of removal of Ca²⁺ from the external solution on both the inward and outward currents at early (A) and late stages (B) of the glucagon response shown in a representative cell (n = 5). CaCl₂ was replaced with MgCl₂. Arrow shows the point when Ca²⁺ was added back to the bath. C, inhibition of the glucagon response by La³⁺ shown in a representative cell (n = 8).

Figure 4. Separation of the currents inhibited by La³⁺

A, time course of La³⁺ block (results similar to those in Fig. 3C are shown using an expanded time scale). Marks 1, 2 and 3 correspond to time points before application of La³⁺, just after application of La³⁺ when only inward current is blocked, and after the complete development of the block, respectively. B, the I–V plots of the currents recorded before addition of La³⁺ (trace 1), at 10 s (trace 2) and at 90 s (trace 3) after La³⁺ addition (cf. corresponding numbers in A). Inset shows currents around the reversal potential. C, the I–V plot of the current blocked by La³⁺ within first 5–10 s (obtained by subtraction of trace 2 from trace 1 shown in B). D, the same current as in C, but recorded in response to a voltage step to −138 mV. E, the I–V plot of the current inhibited by La³⁺ between 10 and 120 s of application (obtained by subtraction of trace 3 from trace 2 shown in B).

Figure 5. Activation of Ba²⁺-permeable channels in rat hepatocytes by glucagon and depletion of intracellular Ca²⁺ stores

A, effect of replacement of the control external solution with solution containing 100 mm Ba²⁺ on the currents activated by glucagon (representative cell, n = 6). B, effect of 100 mm Ba²⁺ on store-operated Ca²⁺ current activated by intracellular perfusion with 20 μm IP₃ (representative cell, n = 9). C, effect of 100 mm Ba²⁺on store-operated Ca²⁺ current activated by 1 μm thapsigargin (representative cell, n = 3). Note absence of the effect when Ba²⁺ was applied before thapsigargin.

Figure 6. Cl⁻ conductance activated by glucagon is inhibited by hepatocyte shrinkage

A, effect of addition of 100 mm sucrose to the external solution on the Cl⁻ currents activated by glucagon. B and C show membrane currents recorded in response to voltage steps ranging between −98 and +82 mV in 20 mV increments in isotonic and hypertonic solutions, respectively. Results shown in a representative cell (n = 4).

Figure 7. Hepatocyte swelling activates Cl⁻ current similar to that activated by glucagon

A, time course of Cl⁻ current development in hypotonic solution and inhibition upon return to control bath solution. Initially, cell was perfused with isotonic solution in which 50 mm NaCl was replaced with 100 mm sucrose, which subsequently was changed to hypotonic solution lacking 100 mm sucrose (see Methods; representative cell, n = 4). B, membrane currents activated by swelling recorded in control solution in response to voltage steps ranging between −98 and +82 mV in 20 mV increments. C, I–V plots of the membrane currents activated by swelling in control solution and in solution in which 140 mm NaCl was replaced with 140 mm sodium glutamate.

Figure 8. Effect of the PLC inhibitor U73122 on the glucagon response

A, U73122 (4 μm) prevents development of the glucagon response (50 nm; n = 4). B, U73343, which is an inactive analogue of U73122, had no effect on the glucagon response. C, U73122 (4 μm) inhibits Cl⁻ current activated by glucagon (representative cell, n = 4). D, U73122 (4 μm) has no effect on Cl⁻ current activated by cell swelling (representative cell, n = 3). E, U73122 (4 μm) has no effect on Ca²⁺ current activated by intracellular perfusion with 20 μm IP₃ (representative cell, n = 3).

Figure 9. Inhibition of PKA-dependent pathway has no effect on glucagon-activated conductance

A, effect of Rp-cAMPS (500 μm in the pipette solution) on glucagon response (50 nm; n = 3). B, effect of PKA inhibitor, H-89 (10 μm), on glucagon response (50 nm; n = 3). Cells were incubated for 20 min with H-89 before application of glucagon.

Figure 10. cAMP activates Cl⁻ conductance in hepatocytes by activating Epac

A, inhibition of the glucagon response by the inhibitor of adenylyl cyclase SQ22536 (n = 4). SQ22536 (0.5 mm) was applied to the bath solution for at least 5 min prior to addition of glucagon. B, effects of cAMP (100 μm in the pipette solution) and its analogues 8-pCPT-2′-O-Me-cAMP (200 μm in the pipette solution) and 6-Bnz-cAMP (200 μm in the pipette solution) on the outward conductance of rat hepatocytes. C, dependence of the cAMP-activated Cl⁻ conductance on intracellular Ca²⁺. Calcium concentration was either weakly buffered to ∼120 nm by 1 mm EGTA, or strongly buffered to ∼10 nm by 10 mm EGTA.