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 t he endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle P achnoda sinuata using in vivo and in vitro methods to measure changes in th e concentration of proline and its precursor alanine. Extracellular calcium is necessary for maximal stimulation of proline synthesis at saturating do ses of Mem-CC (0.3 nM) in vitro. This effect depends on the dose of Ca2+: m aximal proline synthesis of 2.1 mu mol 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 extracellu lar calcium acts. via an influx into the cell, on the stimulation of prolin e production and alanine consumption. The release of calcium from intracell ular sources is part of the signalling process: the agent thapsigargin. whi ch inhibits the Ca2+-ATPase, is able to stimulate proline synthesis in vivo and in vitro. Thimerosal, however, which triggers the release of calcium f rom IP3-sensitive stores in the endoplasmic reticulum. had no influence on proline production nor alanine consumption, indicating that inositolphospha tes are not part of the transduction of the hyperprolinaemic signal of Mem- CC. Both substances, thapsigargin and thimerosal, stimulate calcium entry i n vitro from the medium (similar to Mem-CC), which indicates that a capacit ative calcium entry takes place. Neither the entry of extracellular calcium nor the release from the endoplasmic reticulum, however. are alone suffici ent for a full stimulation of proline synthesis in vitro. The results of th e present study suggest that calcium from extra- as well as from intracellu lar 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. Calciu m acts most likely via the elevation of cAMP levels: the concentration of t his cyclic nucleotide in the fat body during in vitro incubation was elevat ed by 487% by Mem-CC in the presence of calcium, while the increase was onl y 122% when calcium was absent. (C) 2001 Elsevier Science Ltd. All rights r eserved.