Methods for the investigation of neuropeptide catabolism and stability in vitro.

The protocol describes (i) methods for the investigation of neuropeptide catabolism in the central nervous system (CNS), (ii) the identification of the neuropeptidases involved, and (iii) methods for the determination of neuropeptide stability in vitro. These methods are applicable also to study the degradation of peptide hormones by peripheral cells or tissues. To identify peptide degradation products, nanomolar amounts (micromolar concentrations) of peptides are incubated in synthetic media with cell or tissue cultures. Aliquots of the supernatants are withdrawn after different times, peptide fragments separated and fractionated by reversed-phase HPLC, and identified by peptide chemical methods. The peptidases responsible for this degradation can be identified by the use of specific inhibitors listed in the protocol. For receptor binding assays or the study of peptide effects in physiological, nanomolar concentrations the stability of the peptides in an in vitro system should be checked by addition of radiolabeled peptides (femtomolar or nanomolar concentrations) and monitoring the peptide degradation by a procedure analogous to that established for unlabeled peptides. The addition of more or less specific peptidase inhibitors enhances peptide stability in vitro, and thus it can be assured that a given peptide concentration is maintained during biological assays.