Hormonal stimulation of proline synthesis in the fat body of the fruit beetle, Pachnoda sinuata, is calcium dependent

The role of calcium in the transduction of the hyperprolinaemic signal of the endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle Pachnoda sinuata using in vivo and in vitro methods to measure changes in the concentration of proline and its precursor alanine. Extracellular calcium is necessary for maximal stimulation of proline synthesis at saturating doses of Mem-CC (0.3 nM) in vitro. This effect depends on the dose of Ca(2+): maximal proline synthesis of 2.1 micromol mg(-1) protein h(-1) was stimulated by Mem-CC at calcium levels of 0.5 mM, and the EC50 was 0.16 mM. Using the ionophore A 23187 in vivo and in vitro, we demonstrated that the extracellular calcium acts, via an influx into the cell, on the stimulation of proline production and alanine consumption. The release of calcium from intracellular sources is part of the signalling process: the agent thapsigargin, which inhibits the Ca(2+)-ATPase, is able to stimulate proline synthesis in vivo and in vitro. Thimerosal, however, which triggers the release of calcium from IP3-sensitive stores in the endoplasmic reticulum, had no influence on proline production nor alanine consumption, indicating that inositolphosphates are not part of the transduction of the hyperprolinaemic signal of Mem-CC. Both substances, thapsigargin and thimerosal, stimulate calcium entry in vitro from the medium (similar to Mem-CC), which indicates that a capacitative calcium entry takes place. Neither the entry of extracellular calcium nor the release from the endoplasmic reticulum, however, are alone sufficient for a full stimulation of proline synthesis in vitro. The results of the present study suggest that calcium from extra- as well as from intracellular sources is part of the second messenger system for the transduction of the hyperprolinaemic signal of Mem-CC in the fat body of P. sinuata. Calcium acts most likely via the elevation of cAMP levels: the concentration of this cyclic nucleotide in the fat body during in vitro incubation was elevated by 487% by Mem-CC in the presence of calcium, while the increase was only 122% when calcium was absent.