Partial identification of a peptide that stimulates the primary urine production of single isolated Malpighian tubules of the forest ant, Formica polyctena

Abstract

A peptide was purified from a 10% trifluoroacetic acid (TFA) head/thorax extract of 300,000 ants with high performance liquid chromatography (HPLC). Fluid secretion assay of single isolated Malpighian tubules was used as a bioassay. The purity of F. polyctena diuretic peptide (FopDP) after a two step HPLC protocol was confirmed by means of mass spectrometry and revealed a molecular mass of 7514 daltons. Due to lack of material, no enzymatic digestion could be performed and the sequence of only the first 25 amino acids could be determined: VPKYENCVSEVLPAGDRQRCVKVTC. A computer search of sequence data banks did not reveal any significant similarity between FopDP and other known insect diuretic peptides. FopDP had no effect on the basolateral membrane potential and depolarised the apical membrane potential of the Malpighian tubule cells. This effect as well as the stimulatory effect on the primary urine formation in the Malpighian tubule of the ant, could be mimicked with A23187, a calcium ionophore, and by thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. FopDP did not stimulate the cAMP content. The results suggest that FopDP uses an increase of intracellular calcium as cellular transduction mechanism. (C) 1999 Elsevier Science Ltd. All rights reserved.