N-tosyl-l-phenylalanine chloromethyl ketone inhibits lhrh-degrading activity and increases in vitro lhrh release from the immature rat median eminence

Juan Pablo Advis, Ann M. Contijoch, Henryk F. Urbanski, Sergio R. Ojeda

    Research output: Contribution to journalArticle

    7 Scopus citations

    Abstract

    Median eminence (ME) luteinizing-hormone-releasing hormone (LHRH)-degrading activity (LHRH-DA) may play a role in regulating the availability of releasable LHRH. Incubation of LHRH with ME tissue supernatant yields LHRH(1-5) and LHRH(6-10) degradation fragments, as detected by high-performance liquid chromatography (HPLC) analysis, suggesting a 5-6 cleavage of the decapeptide. Since these fragments are also present after incubation of LHRH with α -chymotrypsin (α-CH), we examined the possibility that the irreversible inhibitor of α-CH, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), might inhibit LHRH-DA and affect LHRH release. Irreversible inhibitors of trypsin-like proteases [N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and phenylmethylsulfonylfluoride (PMSF)] were used as controls. LHRH-DA was determined by HPLC estimation of the loss of synthetic LHRH incurred when the peptide was incubated with aliquots of ME supernatant in the presence or absence of the inhibitors. LHRH release from ME fragments was assessed by radioimmunoassay after incubating the tissue with the inhibitors in Krebs-Ringer bicarbonate buffer. The LHRH-DA in both the incubation medium and the ME tissue was determined at the end of the incubation. TPCK (0.5-100 µM) added to ME tissue supernatant inhibited LHRH-DA in a dose-dependent manner. In contrast, when TPCK was added to medium in which intact ME were being incubated to assess LHRH release, the LHRH-DA of these ME was inhibited only at the 25-50-and 100-µM doses of TPCK, suggesting a relative inability of the inhibitor to reach endopeptidase pools in intact tissue. These same doses of TPCK increased LHRH release from the incubated ME. The effect of TPCK (at 50 or 100 µM) on prostaglandin E2 (PGE2) formation was abolished by indomethacin; however, indomethacin blocked only the effect of 50 µMTPCK, but not 100 µMTPCK. On LHRH release. This suggests that at high doses TPCK increases LHRH release through a PGE2-independent mechanism. No evidence of tissue damage after 100 µMTPCK was detected, as assessed by the absence of lactate dehydrogenase levels in the incubation medium. Neither LHRH-DA nor LHRH release were affected by TLCK or PMSF. TPCK at either a low (12.5 µM) or a high (50 µM) dose failed to enhance the effect of norepinephrine on LHRH release, indicating that 5, 6 endopeptidase activity may not act acutely to regulate the pool of releasable LHRH. Our results indicate that (1) TPCK is a potent inhibitor of an endopeptidase which degrades LHRH at its Tyr5-Gly6 bond, and (2) TPCK releases LHRH from the ME not by damaging the nerve terminals or by inhibiting endopeptidase activity, but rather through activation of a cell-membrane-dependent signal transduction mechanism.

    Original languageEnglish (US)
    Pages (from-to)102-108
    Number of pages7
    JournalNeuroendocrinology
    Volume47
    Issue number2
    DOIs
    StatePublished - Jan 1 1988

    Keywords

    • Degradation
    • LHRH
    • Peptidase
    • TPCK

    ASJC Scopus subject areas

    • Endocrinology, Diabetes and Metabolism
    • Endocrinology
    • Endocrine and Autonomic Systems
    • Cellular and Molecular Neuroscience

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