NOREPINEPHRINE-STIMULATED INVITRO RELEASE OF LUTEINIZING-HORMONE-RELEASING HORMONE (LHRH) FROM MEDIAN-EMINENCE TISSUE IS FACILITATED BY INHIBITION OF LHRH-DEGRADING ACTIVITY IN HENS

We and others have previously reported the existence of hypothalamic and anterior pituitary (AP) enzymes that degrade luteinizing hormone (LH)-releasing hormone (LHRH). We have further characterized these LHRH-degrading activities (LHRH-DA) and in addition assessed the role of LHRH-DA in LHRH release from median eminence (ME) tissue in vitro. Major LHRH-DA components were separated and their molecular weights were estimated by gel filtration chromatography. The role of LHRH-DA in LHRH release was determined by release studies from isolated ME, in the presence and absence of N-tosyl L-phenylalanine chloromethyl ketone (TPCK) and/or norepinephrine (NEpi). Degradation and in vitro release studies were performed by using LHRH analogs with amino acid substitutions at their 5-6 bond. Biological activity of these analogs was assessed by measuring in vitro LH release from dispersed anterior pituitary cells. LHRH-DA was determined by high-performance liquid chromatography; LH and LHRH were measured by radioimmunoassay. Separation of LHRH-DA by gel filtration chromatography yielded two major enzymatic activities: a Tyr5-Gly6 cleaving endopeptidase and a post-proline cleaving enzyme. Although LHRH-DA from AP and ME produced identical degradation fragments, the former had 3-fold greater specific activity than the latter. LHRH moieties with a Tyr5-Gly6 bond substitution were more resistant to enzymatic degradation and had greater biological activity than LHRH moieties with a Tyr5-Gly6 bond. TPCK decreased LHRH-DA and increased NEpi-stimulated in vitro release of LHRH from isolated ME. Although these results are consistent with a modulatory role for LHRH-DA on median eminence release of LHRH, further degradation studies are needed during reproductive states in which LHRH release is known to be altered.