Objective: Adrenaline inhibits insulin secretion through activation of a 2 -adrenoceptors (ARs). These receptors are linked to pertussis toxin-sensitive G proteins. Agonist binding leads to inhibition of adenylyl cyclase, inhibition of Ca 2þ channels and activation of K þ channels. Recently, three distinct subtypes of a 2 -AR were described, a 2A -AR, a 2B -AR and a 2C -AR. At present, it is unknown which of these a 2 -AR subtype(s) may regulate insulin secretion. We used mice deficient in a 2 -ARs to analyze the coupling and role of individual a 2 -AR subtypes in insulin-secreting b cells. Methods: The inhibitory effect of adrenaline on insulin secretion was measured in freshly isolated and cultured wild type (wt) and a 2 -AR knockout (KO) mouse islets in order to examine the receptor subtypes which mediate adrenaline-induced inhibition of insulin secretion. Adenylyl cyclase activity was measured in isolated cultured islets. Membrane potential was measured using the amphotericin B permeabilized patch clamp method in isolated and cultured single islet cells. Results: In wt, a 2A -and a 2C -AR KO mouse islets, adrenaline, 1 mmol/l, inhibited secretion by 83, 80 and 100% respectively. In contrast, in a 2A/2C -AR double KO mouse islets, adrenaline had no effect on stimulated secretion indicating that both a 2A -AR and a 2C -AR, but not a 2B -AR, are functionally expressed in mouse islets. Surprisingly, glucose (16.7 mmol/l)-induced secretion in the presence of 1 mmol/l forskolin was greatly impaired in a 2A -AR KO islets. However, when cAMP levels were increased further by the combination of forskolin (5 mmol/l) and 3-isobutyl-1-methylxanthine (100 mmol/l), secretion was stimulated 2.7-fold (8.5-fold in wt islets). Adrenaline lowered the concentration of cAMP in wt and a 2C -AR KO mouse islets by 74%. Adrenaline also hyperpolarized wt and a 2C -AR KO b cells. In contrast, adrenaline did not inhibit adenylyl cyclase in islets of a 2A -AR KO mice, nor did it hyperpolarize a 2A -AR KO b cells. Conclusion: Adrenaline inhibits insulin release through a 2A -and a 2C -ARs via distinct intracellular signaling pathways.

This study examines the effect of epinephrine, a known physiological inhibitor of insulin secretion, on the membrane potential of pancreatic islet cells from sulfonylurea receptor-1 (ABCC8)-null mice (Sur1KO), which lack functional ATP-sensitive K+ (KATP) channels. These channels have been argued to be activated by catecholamines, but epinephrine effectively inhibits insulin secretion in both Sur1KO and wild-type islets and in mice. Isolated Sur1KO beta-cells are depolarized in both low (2.8 mmol/l) and high (16.7 mmol/l) glucose and exhibit Ca(2+)-dependent action potentials. Epinephrine hyperpolarizes Sur1KO beta-cells, inhibiting their spontaneous action potentials. This effect, observed in standard whole cell patches, is abolished by pertussis toxin and blocked by BaCl2. The epinephrine effect is mimicked by clonidine, a selective alpha2-adrenoceptor agonist and inhibited by alpha-yohimbine, an alpha2-antagonist. A selection of K+ channel inhibitors, tetraethylammonium, apamin, dendrotoxin, iberiotoxin, E-4130, chromanol 293B, and tertiapin did not block the epinephrine-induced hyperpolarization. Analysis of whole cell currents revealed an inward conductance of 0.11 +/- 0.04 nS/pF (n = 7) and a TEA-sensitive outward conductance of 0.55 +/- 0.08 nS/pF (n = 7) at -60 and 0 mV, respectively. Guanosine 5'-O-(3-thiotriphosphate) (100 microM) in the patch pipette did not significantly alter these currents or activate novel inward-rectifying K+ currents. We conclude that epinephrine can hyperpolarize beta-cells in the absence of KATP channels via activation of low-conductance BaCl2-sensitive K+ channels that are regulated by pertussis toxin-sensitive G proteins.