Phentolamine Inhibits Exocytosis of Glucagon by Gi2 Protein-dependent Activation of Calcineurin in Rat Pancreatic α-Cells

Marianne Høy,Krister Bokvist,Weng Xiao-Gang,John Hansen,Kirstine Juhl,Per-Olof Berggren,Karsten Buschard,Jesper Gromada

doi:10.1074/jbc.m007562200

Marianne Høy, Krister Bokvist + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m007562200

Copy DOI

Abstract

Capacitance measurements were used to investigate the molecular mechanisms by which imidazoline compounds inhibit glucagon release in rat pancreatic alpha-cells. The imidazoline compound phentolamine reversibly decreased depolarization-evoked exocytosis >80% without affecting the whole-cell Ca(2+) current. During intracellular application through the recording pipette, phentolamine produced a concentration-dependent decrease in the rate of exocytosis (IC(50) = 9.7 microm). Another imidazoline compound, RX871024, exhibited similar effects on exocytosis (IC(50) = 13 microm). These actions were dependent on activation of pertussis toxin-sensitive G(i2) proteins but were not associated with stimulation of ATP-sensitive K(+) channels or adenylate cyclase activity. The inhibitory effect of phentolamine on exocytosis resulted from activation of the protein phosphatase calcineurin and was abolished by cyclosporin A and deltamethrin. Exocytosis was not affected by intracellular application of specific alpha(2), I(1), and I(2) ligands. Phentolamine reduced glucagon release (IC(50) = 1.2 microm) from intact islets by 40%, an effect abolished by pertussis toxin, cyclosporin A, and deltamethrin. These data suggest that imidazoline compounds inhibit glucagon secretion via G(i2)-dependent activation of calcineurin in the pancreatic alpha-cell. The imidazoline binding site is likely to be localized intracellularly and probably closely associated with the secretory granules.

Highlights

Capacitance measurements were used to investigate the molecular mechanisms by which imidazoline compounds inhibit glucagon release in rat pancreatic ␣-cells
Since patients with type 2 diabetes exhibit exaggerated glucagon secretion, our present finding that phentolamine inhibits exocytosis of glucagon may constitute the basis for an additional target for the antidiabetogenic action of this class of compounds
The inhibitory action of phentolamine on exocytosis was not associated with a change in the activity of plasma membrane KATP channels, the activity of voltage-gated Ca2ϩ channels, or changes in cytoplasmic free Ca2ϩ levels but results from a direct interference with the exocytotic machinery, an effect mediated by the protein phosphatase calcineurin

Summary

EXPERIMENTAL PROCEDURES

Preparation of Islets and ␣-Cells—Male Lewis rats (250 –300 g; Møllegaard, Lille Skensved, Denmark) were anesthetized by pentobarbital (100 mg/kg intraperitoneally), and the pancreas was removed. Groups of 10 size-matched rat islets were preincubated for 30 min in 200 ␮l of extracellular solution consisting of 138 NaCl, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 5 mM HEPES (pH 7.4 with NaOH) and 0 –20 mM D-glucose in 96-well Durapore membrane plates (Millipore, Molsheim, France). Statistical significance was evaluated using Student’s t test for paired or unpaired observations or Dunnett’s test for multiple comparisons with a single control

RESULTS

Phentolamine Inhibits Exocytosis Evoked by Intracellular

TABLE I

Clorgyline ϩ phentolamine

DISCUSSION

Phentolamine ϩ pertussis toxin