Differential distribution of isoforms of Leucophaea tachykinin-related peptides (LemTRPs) in endocrine cells and neuronal processes of the cockroach midgut

Five isoforms of tachykinin-related peptides (TRPs), designated LemTRP-1-5, have been identified in the midgut of the cockroach Leucophaea maderae. Th ese peptides have a conserved C-terminus hexapeptide (GFX(1)GX(2)Ramide; X- 1 and X-2 are variable residues) and variable N-termini. Here, we address t he question of whether these five isoforms are all colocalized in the two t ypes of cells in the cockroach midgut, the endocrine cells and the neuronal processes. We also investigate whether the N-terminally extended isoforms LemTRP-2 and -3, which contain putative endoproteolytic cleavage sites, are expressed in intact form or are cleaved in the midgut cells. To this end, we used two approaches. (1) Extracts from portions of the midgut containing each of the cell types were subjected to reverse-phase high performance li quid chromatography (HPLC) and the fractions monitored in a radioimmunoassa y (RIA) with an antiserum to the conserved C-terminus of insect TRPs. (2) A ntisera were raised to the variable N-termini of the extended LemTRP-2 and -3 and used for immunocytochemistry. The HPLC-RIA and immunocytochemical fi ndings indicate that LemTRP-1 and 4-5 are present in the neuronal processes and in endocrine cells of the midgut proper and of the gastric cecae. The two extended forms LemTRP-2 and -3 display a differential distribution: Lem TRP-2 was found in endocrine cells of midgut and gastric cecae, but not in neuronal processes, whereas LemTRP-3 was seen in neuronal processes and end ocrine cells of the midgut proper, but not in the gastric cecae. LemTRP-3 a nd -4 have not been identified in the brain, suggesting further cell- and t issue-specific expression of LemTRPs. The mechanisms behind the cell-specif ic expression of the LemTRPs are not yet understood, but the demonstration of differential distribution of the peptide isoforms provide a first indica tion that the isoforms may have different actions. J. Comp. Neurol. 406:15- 28, 1999. (C) 1999 Wiley-Liss, Inc.