Cellular responses to Pyrularia thionin are mediated by Ca2+ influx and phospholipase A2 activation and are inhibited by thionin tyrosine iodination.

AUTOR(ES)

Evans, J

RESUMO

Pyrularia thionin, isolated from nuts of Pyrularia pubera, is a strongly basic peptide of 47 amino acids. The amino acid sequence and configuration of its four disulfide bonds place this plant peptide, known to be hemolytic, cytotoxic, and neurotoxic, among the thionins. We report and compare several cellular responses mediated by Pyrularia thionin: hemolysis of human erythrocytes, activation of an endogenous phospholipase A2 in Swiss 3T3 cells, cytotoxicity toward HeLa and mouse B16 melanoma cells in culture, viability of rat hepatocytes and lymphocytes measured by trypan blue exclusion, and lethality in mice. Cellular responses related to ion movement include a toxin-mediated influx of Ca2+ into mouse P388 cells measured by Fura-2 fluorescence, depolarization of mouse P388 plasma membrane measured by fluorescence of bis(1,3-diethylthiobarbituric acid)trimethine oxonol (bisoxonol), and depolarization of frog (Rana pipiens) sartorius muscle determined by direct measurement of membrane potential. Graded iodination of Pyrularia thionin leads to a related loss of activity for hemolysis, phospholipase A2 activation, cytotoxicity, and lethality in mice. The mediated Ca2+ influx into and depolarization of P388 cells require Ca2+ in the external medium and are inhibited by 100 microM Ni2+. Depolarization of sartorius muscle by Pyrularia thionin also requires a functional Ca2+ channel, as shown by verapamil inhibition. This muscle depolarization also involves phospholipase A2 activation because dexamethasone and quinacrin, but not indomethacin, protect against depolarization. The IC50 values for viability of rat hepatocytes and splenic lymphocytes measured by trypan blue exclusion were 0.17 and 40 microM, respectively. The general response of cells to Pyrularia thionin involves a membrane alteration leading to depolarization and a channel-mediated influx of Ca2+. There is a related activation of phospholipase A2 that results in loss of membrane integrity, hemolysis in the case of erythrocytes, and eventually cell death. Iodination of Pyrularia thionin leads to a corresponding inhibition of all three cellular responses, which indicates an essential role for tyrosine in either maintenance of peptide structure or interaction of the peptide with cellular membranes.

Documentos Relacionados

• Ca2+ influx and activation of a cation current are coupled to intracellular Ca2+ release in peptidergic neurons of Aplysia californica.
• Coupling of Ca2+ Microdomains to Spatially and Temporally Distinct Cellular Responses by the Tyrosine Kinase Syk*
• Activation of neurohypophysial vasopressin release by Ca2+ influx and intracellular Ca2+ accumulation in the rat.
• Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis.
• Cellular responses to Ca2+ from extracellular and intracellular sources are different as shown by simultaneous measurements of cytosolic Ca2+ and secretion from bovine chromaffin cells.