Protection by serine peptidase inhibitors of endogenous cholecystokinin released from brain slices.
Endogenous cholecystokinin immunoreactivity released by depolarization of slices of rat cerebral cortex undergoes extensive degradation (85% of released immunoreactivity) before reaching the incubation medium. In order to identify the responsible peptidases, a large number of inhibitors of the four catalytic classes were tested for their protective effects. Inhibitors of metallopeptidases (bestatin, amastatin, puromycin, Thiorphan, captopril, o-phenantroline), thiol-peptidases, (leupeptin, antipain, p-hydroxymercuribenzoate) or carboxyl-peptidases (pepstatin) had generally low if any protective effect. By contrast, several serine peptidase inhibitors, i.e. diisopropyl-fluorophosphate, phenylmethylsulphonylfluoride or the chloromethylketone Ala-Ala-Pro-Val-CH2Cl, doubled the recovery of cholecystokinin immunoreactivity and the effect was amplified in the co-presence of bestatin, an aminopeptidase inhibitor and/or Thiorphan, an enkephalinase inhibitor. High-performance liquid chromatographic analysis of the cholecystokinin immunoreactivity recovered in medium in the absence of any inhibitor showed cholecystokinin-8 to be the major peak, representing 8% of the released immunoreactive material. Non-sulphated cholecystokinin-8 represented less than 1%, indicating that desulphation does not constitute a major inactivation pathway for the endogenous octapeptide. Cholecystokinin-5 was the major clearly identifiable immunoreactive fragment, representing 9% of released immunoreactivity in the absence of inhibitors. Its formation was decreased by about 50% in the presence of either diisopropyl-fluorophosphate or bestatin and Thiorphan and abolished when they were associated, suggesting that it resulted from the actions of a serine peptidase(s) and an aminopeptidase(s). Cholecystokinin-6 (or cholecystokinin-7) was less abundant, representing 4% of the released immunoreactivity, and its level was augmented in the presence of diisopropyl-fluorophosphate. Hence a serine endopeptidase cleaving the Met3-Gly4 bond of cholecystokinin-8 may represent a major inactivating peptidase for the endogenous neuropeptide. Additional metabolic pathways not blocked by serine peptidase inhibitors and resulting in the formation of cholecystokinin-6 (or cholecystokinin-7) and, possibly, cholecystokinin-4, are also suggested by the present approach.
Rose
C
C
Unité de Neurobiologie et Pharmacologie (U. 109) de l'INSERM, Centre Paul Broca, Paris, France.
Camus
A
A
Schwartz
J C
JC
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002766:Cholecystokinin / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000494:pharmacology*; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012697:Serine Endopeptidases / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D013450:Sulfones; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D002766:Cholecystokinin; D012697:Serine Endopeptidases; D011188:Potassium