Permissive role of cAMP in the oscillatory Ca2+ response to inositol 1,4,5-trisphosphate in rat hepatocytes

Abstract

Rat hepatocytes respond to alpha-adrenergic stimulation by intracellular production of myo-inositol 1,4,5-trisphosphate (IP3) which stimulates the periodic release and reuptake of intracellular store (IS) Ca2+. The generation of these Ca2+ oscillations was investigated by simultaneously monitoring Ca2+ changes in the cytosol and IS by combined fluorescence microscopy and whole-cell patch clamp. Intracellular IP3 perfusion (1-50 microM in the pipette) produced three types of Ca2+ response: understimulation, oscillations and overstimulation, i.e. with Ca2+ levels not returning to baseline. In a total of 57 experiments, only three displayed oscillations during continuous IP3 infusion, in a narrow range of IP3 concentration centred around 5-8 microM in the pipette. In oscillating cells, cytosolic Ca2+ spikes were synchronized with transient Ca2+ depletions of the IS, consistent with a direct exchange of Ca2+ between the two compartments. Application of 8-Br-cAMP to cells infused with IP3 increased the probability of eliciting Ca2+ oscillations by a factor of 4-5 for IP3 concentrations in the range 1-10 microM, whereas IP3 concentrations above 10 microM always resulted in overstimulation. IP3 photorelease experiments and measurements of IS Ca2+ content indicated that 8-Br-cAMP enhanced the affinity of the IP3 receptor and increased the pool of releasable Ca2+. We propose that cAMP has a permissive role in the generation of IP3-induced Ca2+ oscillations by extending the window of IP3 concentrations able to elicit oscillations.