Mairead M.T. O'Hare

Signal Epitopes in the Three‐Dimensional Structure of Neuropeptide Y

It is generally appreciated that regulatory peptides are recognized as three-dimensional structures by the receptors on their target cells. However, our real knowledge of the relationship between the structure and the function of biologically active peptides to a large degree is limited to some information on the importance of certain parts of their primary structure. Although crystal structures are available for a few peptides, for example glucagon, the relevance of these structures for the biologically active conformation of the peptide is unclear because most peptides do not hold a well-defined structure in aqueous solution. Nevertheless, some of these peptides have been shown by nuclear magnetic resonance (NMR) to fold into ordered conformations when the water activity is decreased by organic solvents or in the presence of lipid micelles. Whether these ‘artificial’ solution structures are of relevance for the receptor-binding conformation of the peptides is an interesting possibility but is still only speculation. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) are members of the so-called PP-fold family of peptides, which differ from most other small

Gastrin releasing peptide in the rat suprachiasmatic nucleus: An immunohistochemical, chromatographic and radioimmunological study

The localization of gastrin releasing peptide-immunoreactive neurons within the rat suprachiasmatic nucleus was investigated with the indirect peroxidase-antiperoxidase and avidin-biotin techniques. Gastrin releasing peptide-immunoreactive perikarya were found in the ventral part of the suprachiasmatic nucleus, which suggests a direct afferent innervation from the retina or the lateral geniculate, placing the gastrin releasing peptide neurons centrally in the regulation of circadian rhythmicity. Gastrin releasing peptide neurons gave rise to fibers and terminals within the suprachiasmatic nucleus, especially in the ventral part among the immunoreactive and non-immunoreactive neurons. The largest number of gastrin releasing peptide-immunoreactive axons leaving the suprachiasmatic nucleus could be followed in a caudodorsal direction to the subparaventricular zone and the dorsal hypothalamic area. Moreover, minor projections could be traced from the suprachiasmatic nucleus rostrally along the dorsal surface of the optic chiasm to the prechiasmatic area, rostrodorsally into the periventricular area and laterally along the dorsal surface of the optic chiasm and tract. Gel filtration and high performance liquid chromatography profiles of the tissue extracts from the suprachiasmatic area revealed the presence of two GRP-immunoreactive peptides, co-eluting with synthetic gastrin releasing peptide18-27 and gastrin releasing peptide1-27 in almost equivalent concentrations. Gastrin releasing peptide18-27 or gastrin releasing peptide1-27 might therefore play a role in regulation of circadian rhythms in hypothalamic nuclei generated by the suprachiasmatic nucleus.

Receptors on phaeochromocytoma cells for two members of the PP‐fold family — NPY and PP

Pancreatic polypeptide (PP) and neuropeptide Y (NPY) belong to a family of regulatory peptides which hold a distinct tertiary structure, the PP‐fold, even in dilute aqueous solution. High‐affinity receptors, specific for both PP and NPY, are described on the rat phaeochromocytoma cell line, PC‐12. The binding of [125I‐Tyr36]PP to PC‐12 cells was inhibited by concentrations of unlabeled PP which correspond to physiological concentrations of the hormone, 10−11‐10−9 mol/1. The affinity of the receptor for the neuropeptide, NPY, was 102‐times lower than that of the PP receptor. C‐terminal fragments of both PP (PP24–36) and NPY (NPY13–36) were between 102 ‐ and 103‐times less potent in displacing the radiolabeled 36‐amino‐acid peptides from their respective receptors. It is concluded that PC‐12 cells are suited for structure‐function studies of the PP‐fold peptides and studies on the cellular events following cellular binding of PP‐fold peptides.

Neuropeptide Y in guinea pig, rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17.

Neuropeptide Y (NPY) was isolated and characterised from acid-ethanol extracts of rabbit and guinea pig brain. In both instances the chromatographic purification was a two-step procedure of gel filtration followed by reverse-phase high-performance liquid chromatography. The amino acid sequence of rabbit and guinea pig NPY was found to be identical to human and rat NPY as deduced from the cDNA structures. With the exception of the porcine peptide, all mammalian NPYs characterised to date have a methionine residue in position 17. This methionine residue is readily oxidized as indicated by the high degree of spontaneous oxidation of peptides found in the rabbit and guinea pig brain extracts and in NPY extracted from a rat phaeochromocytoma cell line. It is concluded that NPY is among the most highly conserved peptides and that NPYs containing methionine in position 17 are prone to oxidation.

An immunohistochemical and chromatographic analysis of the distribution and processing of proneuropeptide Y in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) regulates a number of circadian rhythms in mammals. A neuropeptide Y (NPY)-containing pathway from the intergeniculate leaflet of the lateral geniculate to the SCN is considered to carry information of the environmental light-dark cycle. Antisera directed against NPY, Cys-NPY(32-36)amide or the C-terminal extended peptide of proNPY(68-97) (CPON) and avidin-biotin immunohistochemistry were used to define the precise distribution of NPYergic nerve fibers in the SCN, and to compare the location of the various fragments of proNPY in these nerves. Gel chromatography and specific radioimmunoassays were applied to quantify the efficiency of the amidation of NPY, and to study the size of peptides demonstrating NPY- and NPYamide-immunoreactivity in anterior hypothalamic extracts. NPY-, NPYamide-, and CPON-immunoreactive nerve fibers exhibited apparently the same distribution and morphology in the SCN. Immunoreactive fibers were preferentially located in the ventral part of the SCN, but along the rostrocaudal axis of the nucleus, the density and the precise distribution of immunoreactive elements changed. From the rostral third of the SCN to the middle third, the number of immunoreactive fibers increased and their distribution extended in a dorsal and lateral direction. In the caudal part of the SCN, the number of immunoreactive elements decreased and the innervation spread to an even more dorsolateral location. Dorsal aspects of the rostral SCN contained a moderate number of fibers, whereas the dorsomedial quadrant of the caudal 2/3 of the SCN was almost devoid of immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Regional Distribution of Neuropeptide Y and Its Receptor in the Porcine Central Nervous System

Abstract: The regional distribution of neuropeptide Y (NPY) immunoreactivity and receptor binding was studied in the porcine CNS. The highest amounts of immunoreactive NPY were found in the hypothalamus, septum pellucidum, gyrus cinguli, cortex frontalis, parietalis, and piriformis, corpus amygdaloideum, and bulbus olfactorius (200–1,000 pmol/g wet weight). In the cortex temporalis and occipitalis, striatum, hippocampus, tractus olfactorius, corpus mamillare, thalamus, and globus pallidus, the NPY content was 50–200 pmol/g wet weight, whereas the striatum, colliculi, substantia nigra, cerebellum, pons, medulla oblongata, and medulla spinalis contained <50 pmol/g wet weight. The receptor binding of NPY was highest in the hippocampus, corpus fornicis, corpus amygdaloideum, nucleus accumbens, and neurohypophysis, with a range of 1.0–5.87 pmol/mg of protein. Intermediate binding (0.5–1.0 pmol/mg of protein) was found in the septum pellucidum, columna fornicis, corpus mamillare, cortex piriformis, gyrus cinguli, striatum, substantia grisea centralis, substantia nigra, and cerebellum. In the corpus callosum, basal ganglia, corpus pineale, colliculi, corpus geniculatum mediale, nucleus ruber, pons, medulla oblongata, and medulla spinalis, receptor binding of NPY was detectable but <0.5 pmol/mg of protein. No binding was observed in the bulbus and tractus olfactorius and adenohypophysis. In conclusion, immunoreactive NPY and its receptors are widespread in the porcine CNS, with predominant location in the limbic system, olfactory system, hypothalamoneurohypophysial tract, corpus striatum, and cerebral cortex.

Partial processing of the neuropeptide Y precursor in transfected CHO cells

The activation of regulatory peptides by post‐translational modification of their biosynthetic precursors is generally thought to occur only in neuroendocrine cells. We have selected clones of Chinese hamster ovary cells, a non‐neuroendocrine cell line, which were transfected with a eukaryotic expression vector coding for the precursor for neuropeptide Y. Although the majority of the immunoreactive NPY was found in the form of pro‐NPY, some degree of intracellular proteolytic processing of the precursor occurred in all clones. Part of the intracellular NPYimmunoreactivity was even correctly amidated. Extracellular degradation of pro‐NPY in the tissue culture medium generated immunoreactivity which corresponded in size to NPY. It is concluded that precursor processing can occur in non‐neuroendocrine cells both as a biological process within the cells and as apparent processing, degradation in the tissue culture medium.

Interaction of NPY and VIP in regulation of myometrial blood flow and mechanical activity.

The occurrence, molecular characteristics and biological function of neuropeptide Y (NPY) has been studied in the female genital tract of non-pregnant rabbits. NPY immunoreactivity was demonstrated throughout the genital tract. Maximum concentrations were found in the salpinx (fallopian tube), 570 pmol/g (median) lower within the uterine body (1.5 pmol/g), cervix (2.8 pmol/g) and vagina (3.6 pmol/g). In vitro, NPY had a dose-dependent stimulatory effect on non-vascular smooth muscle (ED50 10(-9) mol/l) as studied by myometrial tension recordings. In vivo, NPY (50 pmol/min.kg) induced a dose-related, non-adrenergic and non-cholinergic decrease in myometrial blood flow. Small C-terminal (NPY31-36) or N-terminal (NPY1-16) fragments of NPY had no effect on myometrial blood flow. NPY was found to interact with the smooth muscle effect of VIP; the presence of VIP (10(-8) mol/l) counteracted the contraction elicited by NPY (10(-8) mol/l) returning the response to control value. VIP and NPY displayed a similar physiological antagonism on myometrial blood flow. There was a clear difference in the response to VIP and NPY as the effect of NPY on myometrial blood flow first appeared after a lag period of 2 minutes whereas the effect of VIP was almost instantaneous. It is concluded that NPY and VIP may interact in the local nervous control of genital functions.

Gastrin releasing peptide (GRP) is present in a GRP(1–27) form in anterior pituitary cells of the guinea pig

Immunohistochemical and chromatographic studies were performed on the guinea pig anterior pituitary gland with an antiserum recognizing an epitope within the gastrin releasing peptide (GRP) carboxyterminal amino acid sequence Val-Gly-His-Leu-Met-NH2. Within the anterior pituitary gland GRP-like immunoreactive cells were identified. The GRP-like immunoreactive cells were distributed heterogenously in the gland, predominantly located in ventral aspects of the anterior pituitary. Intracellularly, the immunoreactivity elements were identified as granula-like structures in the cytoplasma. To further characterize the peptide displaying GRP-like immunoreactivity within the pituitary cells, the GRP-like substances were analyzed by radioimmunoassay and gel filtration chromatography. Using this analytical approach it was determined that the guinea pig pituitary extract contained a peptide with characteristics similar to that of authentic porcine GRP(1-27). Only trace amounts of smaller C-terminal fragments were identified. These results indicate, in contrast to findings in other tissues, the GRP(1-27) is not further degraded into smaller peptide fragments.

Immunohistochemical and chromatographic identification of peptides derived from proneuropeptide Y in the human frontal cortex

Proneuropeptide Y (proNPY) is posttranslationally processed to NPY(1-36)amide and the C-terminal flanking peptide of NPY (CPON). Antisera directed against the N-terminal part of NPY, CPON, or CysNPY(32-36)amide were used to identify peptide fragments processed from proNPY in biopsies of human frontal cortical specimens obtained from patients who underwent surgical treatment of profound cerebral tumors. Gel filtration and radioimmunoassays of human cortical extracts revealed that the NPY immunoreactivity was found only as NPY(1-36)amide, indicating that all NPY is present in an amidated form. In contrast, no intact proNPY was identified. NPY/CPON-immunoreactive neurons were observed to be nonspiny with long axonal processes mostly orientated longitudinally in the direction of the superficial layers. Bundles of immunoreactive fibers in the underlying white matter were orientated toward superficial layers of the neocortex, indicating a subcortical origin of some NPY/CPON nerve fibers. Axonal terminals were distributed throughout the neocortex, with highest numbers observed in layer I. Some fibers penetrated from the superficial layer I into the overlying pial surface. Many fibers were also observed in proximity to intracortical blood vessels, and some of these fibers originated from the cortical neurons, indicating that NPY could play a role as an intracortical autoregulator of the tonus of cerebral arterioles. Together these results indicate that NPY(1-36)amide and CPON are present in intracortical neurons as two independent molecules and that NPY may be involved in synaptic processes and regulation of blood flow in the human brain.