dynorphins and 1-10-phenanthroline

The effect of peptidase inhibitors on the degradation of [3H]-bradykinin by rat hypothalamic slices was studied using HPLC to separate and identify the products. The degradation appears to be mainly mediated by an enzyme which cleaves the peptide at the Phe5-Ser6 bond and is inhibited by 1,10-phenanthroline, dynorphin(1-13) and carboxyphenylethyl-Ala-Ala-Phe-p-aminobenzoate. This suggest the involvement of a membrane bound variant of the soluble metalloendopeptidase (EC3.4.24.15) isolated from rat brain which degrades neurotensin, angiotensin and other neuropeptides as well as bradykinin.

Topics: 4-Aminobenzoic Acid; Animals; Bradykinin; Captopril; Chromatography, High Pressure Liquid; Dynorphins; Endopeptidases; Glycopeptides; Hypothalamus; Leucine; Male; Metalloendopeptidases; Neurotensin; Oligopeptides; para-Aminobenzoates; Peptide Fragments; Phenanthrolines; Rats; Rats, Inbred Strains

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.

Topics: Adenosine Triphosphate; Adrenal Medulla; Amino Acid Sequence; Animals; Bacitracin; Cattle; Dynorphins; Endorphins; Enkephalin, Leucine; Enkephalin, Methionine; Epinephrine; Guinea Pigs; Leucine; Nicotine; Norepinephrine; Peptide Fragments; Phenanthrolines; Rabbits; Receptors, Opioid; Thiorphan; Tiopronin