Neuropeptide degradation produces functional inactivation in the crustacean nervous system

Abstract

The pentapeptide proctolin (Proct.; Arg-Tyr-Leu-Pro-Thr) is a modulatory transmitter found throughout the crustacean nervous system. No information is available in this system, however, as to how the actions of this peptide are terminated. To study this issue in the crab Cancer borealis, we incubated exogenous proctolin (10(-5) M) with either the thoracic ganglion (TG) or with conditioned saline (CS) that had been preincubated with the TG. We removed aliquots at standard time points for analysis by reverse-phase high-performance liquid chromatography (HPLC). We found that over time the proctolin peak became progressively smaller, while three novel peaks appeared and increased in size. Comigration experiments using HPLC indicated that the major novel peak was Proct. (Tyr-Leu-Pro-Thr), while one of the two minor peaks was Proct. (Leu-Pro-Thr). The other minor peak appeared to be Proct. (Arg-Tyr), based on similar HPLC retention time to synthetic Proct. The reduction in the proctolin peak and the increase in the Proct. peak was prevented by co-incubation of proctolin with any one of several aminopeptidase inhibitors (10(-4) M). Proct. and Proct. appeared to result from a diaminopeptidase-mediated cleavage of proctolin. We tested whether N-terminal cleavage functionally inactivated proctolin by coapplying proctolin (10(-8) M) and individual aminopeptidase inhibitors (10(-5) M) to the isolated stomatogastric ganglion (STG). We found that these inhibitors significantly enhanced the proctolin excitation of the pyloric rhythm. Furthermore, application of synthetic Proct. to the STG had no effect unless high concentrations (> 10(-6) M) were used, and neither Proct. nor Proct. (10(-4) M) influenced the pyloric rhythm. Our results indicate that proctolin is enzymatically degraded and thereby biologically inactivated in the crab nervous system, primarily by extracellularly located aminopeptidase activity.

Neuropeptide signaling near and far: how localized and timed is the action of neuropeptides in brain circuits?

“…Application of peptidase inhibitors strengthens the action of both bath applied and neuronally released peptides. Effects can also be seen in rhythmic network activity (Coleman et al 1994;Nusbaum 2002;Wood and Nusbaum 2002). Thus, it is suggestive that membrane bound peptidases play an important role in limiting the spread and temporal action of neuropeptides in the neuropil of a ganglion.…”

“…One cell body is indicated (cb). a-d, g Are previously unpublished, e is from Skaer et al (2002)and f is from Nässel (2002) (STG) of a crab by application of peptidase inhibitors (Coleman et al 1994;Nusbaum 2002;Stein et al 2007). Specific blocking of peptidases known to inactivate either crab tachykinin or proctolin produces dramatic effects on network activity in the STG.…”

Proctolin in the post-genomic era: new insights and challenges

“…Direct evidence in support of a functional role for an aminopeptidase in affecting the output of proctolinergic neurons has been obtained by studying the effect of aminopeptidase inhibitors on the actions of proctolin in the modulation of the pyloric rhythm by the stomatogastric nervous system in the crab, C. borealis (Coleman et al 1994;Wood and Nusbaum 2002). Both an aminopeptidase and a DPP-3-like enzyme were identified as proctolin-inactivating peptidases in the CNS of C. borealis with the former peptidase dominating the metabolism.…”

“…The metabolism and inactivation of proctolin in insects and crustaceans has been studied in several laboratories, identifying several proctolin-degrading peptidases (Quistad et al 1984;Starratt and Steele 1984;Isaac 1987;Puiroux and Loughton 1992;Coleman et al 1994). These enzymes were, however, not characterised at the molecular level and their precise identity remained unknown.…”

Proline-specific dipeptidyl peptidase activity in the cockroach brain and intestine: Partial characterization, distribution, and inactivation of tachykinin-related peptides

“…It has also been shown that the peptidase activity for degrading both neuropeptides and hormonal peptides may be bound to the surface of the target tissues in arthropods (Rayne and O'Shea, 1992;Coleman et al, 1994;Li et al, 1997). Thus, we tested the duration of the myostimulatory response of LemTRP-1 and the blowfly peptide CavTK-I on the L. maderae hindgut when applied with and without peptidase inhibitors.…”