Neuropeptide Y1-36 (NPY1-36) acts through Y1 and Y2 receptors while the C-terminal NPY fragments NPY18-36 and N-acetyl[Leu28,31]pNPY24-36 act only through the Y2 receptor. We have investigated the effects of intracerebroventricular (i.c.v.) administration of NPY1-36, NPY18-36 and N-acetyl[Leu28,31]pNPY24-36 on LH secretion in the ovariectomised (OVX) ewe. These peptides were administered into a lateral ventricle (LV) or the third ventricle (3V) of OVX ewes during the non-breeding and breeding seasons. Microinjections of NPY were also made into the preoptic area (POA) during both seasons to investigate the effects of NPY at the level of the GnRH cell bodies. Tamed sheep were fitted with 19 gauge guide tubes into the LV, 3V or the septo-preoptic area (POA). Jugular venous blood samples were taken every 10 min for 3 h. Sheep were then given NPY1-36 (10 micrograms), NPY18-36 (100 micrograms) or saline vehicle into the LV; N-acetyl[Leu28,31]pNPY24-36 (100 micrograms), NPY1-36 (10 micrograms or 100 micrograms), NPY18-36 (10 micrograms or 100 micrograms) or saline vehicle into the 3V, or NPY1-36 (1 microgram, 5 micrograms, 10 micrograms) into the POA. Blood sampling continued for a further 3 h. LH was measured in plasma by radioimmunoassay. LV or 3V injection of 10 micrograms NPY1-36 caused a small but significant (P < 0.025) increase in the interval from the last pre-injection pulse of LH to the first post-injection LH pulse during the breeding season. Other LH pulse parameters were not significantly affected. NPY18-36 did not produce any significant change in LH pulsatility when injected into the LV, and neither peptide had any effect on plasma prolactin or GH levels. There was a significant (P < 0.01) reduction in LH pulse frequency after 3V injection of 10 micrograms and 100 micrograms NPY and 100 micrograms NPY18-36. Pulse amplitude was reduced by 3V administration of the Y2 agonist, N-acetyl[Leu28-31]pNPY24-36 and 100 micrograms NPY18-36. When the amplitude of the first post-injection LH pulse was analysed, 10 micrograms NPY also had a significant (P < 0.05) suppressive effect. During the non-breeding season, 100 micrograms NPY1-36 (but not 10 micrograms) decreased (P < 0.01) LH pulse frequency. LH pulse amplitude was significantly (P < 0.01) decreased by 100 micrograms NPY18-36. Doses of 10 micrograms NPY1-36 and 100 micrograms NPY18-36 had greater inhibitory effects on pulse frequency during the breeding season but the suppressive effect of 100 micrograms NPY was similar between seasons. Microinjections of NPY into the POA decreased (P < 0.01) average plasma LH levels during the non-breeding season at a dose of 10 micrograms but did not significantly affect pulse frequency or amplitude. We conclude that a substantial component of the inhibitory action of NPY on LH secretion in the absence of steroids is mediated by the Y2 receptor. This inhibition is probably exerted by way of a presynaptic action on GnRH terminals in the median eminence as NPY does not modulate the frequency or amplitude of LH pulses at the lev...
In an effort to gain insight into the bioactive conformation of neuropeptide Y upon interaction with its receptors, all single-point D-amino acid substituted NPY analogues were prepared, and their Y1 and Y2 receptor binding affinities were evaluated using the human neuroblastoma cell lines, SK-N-MC and SK-N-BE2, respectively. Solid-phase synthesis (Boc strategy) followed by preparative HPLC purification produced analogues of high purity that were characterized by RP-HPLC, AAA, LSIMS, CZE, and optical rotation. Of the 37 isomers (a naturally occurring glycine at position 9 was replaced by Ala and D-Ala), Y1 receptor binding was most perturbed by chiral inversion of residues at the C-terminus (residues 20, 27, 29-35, Ki > or = 300 nM). Substitutions at residues 2-5, 28, and 36 had Ki values ranging from 40 to 260 nM. Substitutions at all other positions yielded analogues with affinities ranging from 1.5 to 20 nM. Binding affinities to the Y2 class of receptors all measured in the low or sub-nanomolar concentrations, with the exception of C-terminally modified isomers (residues 30-35). Only [D-Arg33]- and [D-Gln34]NPY displayed no measurable binding affinity to Y2 receptors at the highest concentration tested (1000 nM). Representative analogues were selected on the basis of their binding affinities and position in the sequence for structural analysis using circular dichroism (CD) spectroscopy. Of the nine peptide evaluated ([D-Pro5]-, [Ala9]-, [D-Glu10]-, [D-Asp11]-, [D-Ala18]-, [D-Tyr20]-, [D-Tyr27]-, and [D-Arg33]NPY), only [D-Tyr27]NPY expressed a definitive correlation between loss of binding affinity and disruption of secondary structure by having the propensity to form beta-sheets at the expense of alpha-helical content. It was concluded that although the incorporation of a single D-amino acid within the sequence of NPY may confer a conformational perturbation, the receptor interaction was only affected when certain critical residues were modified, findings that provide a basis for the identification of the binding pharmacophore of NPY.
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