1. Effect of [D-Trp6]LHRH infusion on prolactin secretion by perifused rat pituitary cells
I Torres-Aleman, M Fernández, L Debeljuk, A L Charro Regul Pept. 1987 Jul;18(1):19-28. doi: 10.1016/0167-0115(87)90046-2.
The effect of a superactive agonistic analog of luteinizing hormone-releasing hormone (LHRH), [D-Trp6]LHRH on prolactin (PRL) secretion by perifused rat pituitary cells was investigated. Constant infusion of [D-Trp6]LHRH (0.5 ng/min) for 2-3 h elicited a significant decrease in PRL secretion by these cells. This decrease in PRL release started ca. 30 min after the beginning of the infusion with the LHRH analog and lasted up to 1.5-2 h. [D-Trp6]LHRH significantly stimulated luteinizing hormone (LH) secretion during the first 30 min of peptide infusion; thereafter, LH levels began to return to control values. In animals pretreated in vivo with 50 micrograms of [D-Trp6]LHRH (s.c.) 1 h before sacrifice, PRL secretion by the rat pituitary cell perifusion system was significantly lower than vehicle-injected controls throughout the entire [D-Trp6]LHRH infusion period. On the other hand, thyrotropin-releasing hormone (TRH)-stimulated PRL secretion was slightly, but significantly imparied by [D-Trp6]LHRH infusion, while dopamine (DA) inhibition of PRL release was unaffected by this same treatment. These results reinforce previous observations of a modulatory effect of [D-Trp6]LHRH, probably mediated by pituitary gonadotrophs, on PRL secretion by the anterior pituitary. In addition, our findings suggest that basal PRL secretion by the lactotroph may be dependent on a normal function of the gonadotroph. The collected data from this and previous reports support the existence of a functional link between gonadotrophs and lactotrophs in the rat pituitary gland.
2. Metabolism of [Des-Gly10,D-Trp6]LHRH ethylamide in rabbit nasal tissue
U B Kompella, B A Dani Life Sci. 1996;58(24):2201-7. doi: 10.1016/0024-3205(96)00214-7.
The objective of this study was to determine whether [Des-Gly10, D-Trp6] LHRH ethylamide, a nonapeptide LHRH agonist known as deslorelin, is degraded by the rabbit nasal tissue. Deslorelin was incubated with nasal tissue either alone or in the presence of 0.1 mM ouabain, 0.1% 2,4-dinitrophenol, 0.1 mM phosphoramidon, 0.1 mM N-tosyl-L-phenylalanine chloromethylketone (TPCK) or 2% EDTA at 37 degrees C. Furthermore, deslorelin alone was incubated with nasal tissue at 4 degrees C. All incubation solutions were adjusted to isotonicity and pH 5.0. At the end of 90 min, the supernatants were analyzed using a reversed-phase HPLC. Metabolite peaks could be detected in all the above experiments except the low temperature study, suggesting inhibition of metabolism at low temperature. Intact drug remaining in the supernatant was elevated by about 32% by ouabain and dinitrophenol, suggesting that energy-dependent cellular uptake is likely for deslorelin. Phosphoramidon and TPCK failed to alter deslorelin levels, suggesting that phosphoramidon and TPCK sensitive endopeptidases did not contribute to the observed deslorelin metabolism. However, EDTA significantly elevated the intact deslorelin levels in a dose-dependent manner, with an elevation of 113% with 2% EDTA. With 2% EDTA, the metabolite peaks almost completely disappeared, indicating a possible role for either metal-activated peptidases or metallo-endopeptidases in the nasal metabolism of deslorelin.
3. Effects of D-amino acid substituents on degradation of LHRH analogues by proximal tubule
G Flouret, T Majewski, D R Peterson, A J Kenny, F A Carone Am J Physiol. 1987 Mar;252(3 Pt 1):E320-6. doi: 10.1152/ajpendo.1987.252.3.E320.
Less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, the luteinizing hormone-releasing hormone, LHRH, is degraded in renal proximal tubules (PT) in vivo (rat) and in vitro (rabbit) to less than Glu-His (2), less than Glu-His-Trp (3), and less than Glu-His-Trp-Ser (4). LHRH may be cleaved by endopeptidases simultaneously at multiple bonds, or initially at Ser4-Tyr5 followed by carboxypeptidase hydrolysis of 4 to 3 and then 2. To distinguish between these mechanisms, [3H]LHRH analogues were incubated with rabbit renal brush-border membranes (BBM), microinfused into PT in vivo or in vitro, and products were analyzed by HPLC. [D-Ser4]LHRH was not cleaved at D Ser4-Tyr5 but yielded less than Glu-His-Trp-D-Ser-Tyr-Gly as the major metabolite plus 2 and 3. [D-Trp6]LHRH was cleaved by BBM and PT to 2 and 3, but not to 4. [D-Ser4, D-Trp6]LHRH was not cleaved by BBM, but was degraded to 2 by PT in vivo. Thus, D-amino acid substituents altered the expected cleavage pattern of these analogues. [3H]LHRH was cleaved by BBM or by endopeptidase-24.11 from porcine PT to metabolites 2, 4, small amounts of 3, and less than Glu-His-Trp-Ser-Tyr-Gly, but cleavage was strongly inhibited by the specific inhibitor phosphoramidon. Thus, normally LHRH may be cleaved in PT by endopeptidase-24.11 to 2 and 4, and by angiotensin I-converting enzyme to 3, its known cleavage site.
