Two novel tachykinin-related peptides from the nervous system of the crab Cancer borealis.

Abstract

Immunocytochemical and biochemical studies have indicated the presence of many neuroactive substances in the stomatogastric nervous system (STNS) of the crab Cancer borealis. In electrophysiological studies, many of these substances modulate the motor output of neural networks contained within this system. Previous work in the STNS suggested the presence of neuropeptides related to the invertebrate tachykinin-related peptide (TRP) family. Here we isolate and characterize two novel peptides from the C. borealis nervous system that show strong amino acid sequence identity to the invertebrate TRPs. The central nervous systems of 160 crabs were extracted in an acidified solvent, after which four reversed-phase HPLC column systems were used to obtain pure peptides. A cockroach hindgut muscle contraction bioassay and a radioimmunoassay (RIA) employing an antiserum to locustatachykinin I (Lom TK I) were used to monitor all collected fractions. The amino acid sequences of the isolated peptides were determined by Edman degradation. Mass spectrometry and chemical synthesis confirmed the sequences to be APSGFLGMR-NH2 and SGFLGMR-NH2. APSGFLGMR-NH2 is approximately 20-fold more abundant in the crab central nervous system than is SGFLGMR-NH2. We have named these peptides Cancer borealis tachykinin-related peptide Ia and Ib (CabTRP Ia and Ib), respectively. Both peptides are myoactive in the cockroach hindgut muscle contraction bioassay, with CabTRP Ia being approximately 500 times more potent than CabTRP Ib. RIA performed on HPLC-separated C. borealis stomatogastric ganglion (STG) extract revealed that CabTRP Ia is the only detectable TRP-like moiety in this ganglion. Incubation of synthetic CabTRP Ia with the isolated STG excited the pyloric motor pattern. These effects were suppressed by the broad-spectrum tachykinin receptor antagonist Spantide I. Spantide I had no effect on the actions of the unrelated endogenous peptide proctolin in the STG. There was no consistent influence of CabTRP Ib on the pyloric rhythm. Given its amino acid sequence and minimal biological activity in the crab, CabTRP Ib may be a breakdown product of CabTRP Ia.