Synergistic Actions of Glucagon and Miniglucagon on Ca ²⁺ Mobilization in Cardiac Cells

Abstract

It has been recently shown that the physiological processing of glucagon into its C-terminal (19-29) fragment, miniglucagon, by cardiac cells was essential for the contractile positive inotropic effect of the hormone. However, the mechanisms underlying the effects of miniglucagon remained undetermined. In the present study, we assessed the effects of miniglucagon on Ca2+ homeostasis in embryonic chick ventricular myocytes. In quiescent cells, short-term applications of 0.1 nmol/L miniglucagon markedly increased the accumulation of 45Ca into intracellular compartments resistant to digitonin lysis and sensitive to caffeine. Ca2+ accumulation into the sarcoplasmic reticular (SR) store was further attested by fura 2 imaging studies on quiescent or prestimulated cells: miniglucagon potentiated Ca2+ release from the SR compartment triggered by caffeine and evoked a rise in cytosolic Ca2+ when applied on cells pretreated with 1 mumol/L thapsigargin, a specific inhibitor of the SR Ca2+ pump. Glucagon alone produced a small cytosolic Ca2+ signal that was considerably amplified by miniglucagon. The action of glucagon was mimicked by 8-bromo-cAMP and was blocked by isradipine, suggesting that it relied on the activation of L-type Ca2+ channels, via phosphorylation. We conclude that the combined actions of miniglucagon and glucagon on Ca2+ accumulation into SR stores and Ca2+ release from the same stores are likely to support the positive inotropic effect elicited in vivo by glucagon on heart contraction.

Evidence for a β2-Adrenergic/Arachidonic Acid Pathway in Ventricular Cardiomyocytes

“…Glucagon action in heart relies on the synergistic actions of glucagon itself and its metabolite (19 -29), mini-glucagon (15,22). We have demonstrated that cAMP mediates glucagon action and that AA is the second messenger of mini-glucagon (15).…”

“…Primary monolayer cultured heart cells were prepared from 13-day-old chick embryo ventricles as described previously (15,21,22). Briefly, cells were dissociated by repeated cycles of trypsinization.…”

“…Cells were then washed with saline buffer B (2 ϫ 2 ml) and allowed to incubate in the same buffer for 15 min at 25°C to facilitate hydrolysis of intracellular Fura-2/AM. The concentration of Fura-2 in myocytes was estimated as described previously (15,21,22), according to the procedure of Donnadieu et al (23). Under usual loading conditions, the average intracellular concentration of Fura-2 was 15 M. Ca 2ϩ imaging, developed by A. Trautman in collaboration with the IMSTAR (Paris, France), was essentially as described by Sauvadet et al (21).…”

β2-Adrenergic Receptor Agonists Increase Intracellular Free Ca2+ Concentration Cycling in Ventricular Cardiomyocytes through p38 and p42/44 MAPK-mediated Cytosolic Phospholipase A2 Activation

“…Primary monolayer cultured heart cells were prepared from 13-day-old embryonic chick ventricles as previously described (5,6,21,22). Briefly, cells were dissociated by repeated cycles of trypsinization.…”

“…Cells were then washed two times with saline buffer B and allowed to incubate in the same buffer for 15 min at 25°C to facilitate hydrolysis of intracellular Fura-2/AM. The concentration of Fura-2 in myocytes was estimated as described previously (5,6,21,22) according to the procedure of Donnadieu et al (23). Under usual loading conditions, the average intracellular concentration of Fura-2 was 15 M. Ca 2ϩ imaging was essentially performed as described by Sauvadet et al (6,21).…”