Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.

Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5μg/kg dose administered 1h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P<0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y(1), a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y(1) receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y(1) found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells.

Novel dimeric DOTA-coupled peptidic Y1-receptor antagonists for targeting of neuropeptide Y receptor-expressing cancers

ABSTRACT:

Amelioration of portal hypertension and the hyperdynamic circulatory syndrome in cirrhotic rats by neuropeptide Y via pronounced splanchnic vasoaction

Splanchnic vasodilation triggers the development of the hyperdynamic circulatory syndrome in portal hypertension. Neuropeptide Y (NPY), a sympathetic co-transmitter of norepinephrine, improves contractility in mesenteric arteries of pre-hepatic portal hypertensive rats. Therefore, we investigated the effect of NPY on mesenteric arterial contractility in vitro and in vivo in cirrhotic ascitic rats, as well as the vasoactive pathways involved.

No association of the neuropeptide Y (Leu7Pro) and ghrelin gene (Arg51Gln, Leu72Met, Gln90Leu) single nucleotide polymorphisms with eating disorders

Genetic factors likely contribute to the biological vulnerability of eating disorders.

Dysbalance in sympathetic neurotransmitter release and action in cirrhotic rats: impact of exogenous neuropeptide Y

Splanchnic vasodilation is an essential disturbance in portal hypertension. Increased systemic sympathetic nerve activity is well known, but potential corresponding vascular desensitization is incompletely characterized. Release of splanchnic sympathetic neurotransmitters noradrenaline (NA) and co-transmitter neuropeptide Y (NPY) remains to be elucidated. Finally, the effects of exogenous NPY on these mechanisms are unexplored.

Brain levels of neuropeptide Y in experimental pneumococcal meningitis

Neuropeptide Y (NPY), which is found in high concentrations in several regions of the brain including nuclei of the brain stem and in nerve fibers surrounding cerebral vessels, has been proposed to play a role in regulating cerebral blood flow (CBF) and systemic vegetative functions. Since CBF is altered during meningitis, we examined whether NPY concentrations changed in various regions of the rabbit brain in response to experimental pneumococcal meningitis. Changes were most pronounced in the medulla, where NPY concentration increased threefold after 48 h of infection. Concomitantly, there was an increase in NPY immunoreactive fibers surrounding small vessels in the dorsolateral medulla, especially in the nucleus tractus solitarius. These results suggest that NPY may play a role in inducing some of the hemodynamic changes seen during pneumococcal meningitis.

High expression of neuropeptide Y1 receptors in ewing sarcoma tumors

PURPOSE: Peptide receptors are frequently overexpressed in human tumors, allowing receptor-targeted scintigraphic imaging and therapy with radiolabeled peptide analogues. Neuropeptide Y (NPY) receptors are new candidates for these applications, based on their high expression in specific cancers. Because NPY receptors are expressed in selected sarcoma cell lines and because novel treatment options are needed for sarcomas, this study assessed the NPY receptor in primary human sarcomas. EXPERIMENTAL DESIGN: Tumor tissues of 88 cases, including Ewing sarcoma family of tumors (ESFT), synovial sarcomas, osteosarcomas, chondrosarcomas, liposarcomas, angiosarcomas, rhabdomyosarcomas, leiomyosarcomas, and desmoid tumors, were investigated for NPY receptor protein with in vitro receptor autoradiography using (125)I-labeled NPY receptor ligands and for NPY receptor mRNA expression with in situ hybridization. RESULTS: ESFT expressed the NPY receptor subtype Y1 on tumor cells in remarkably high incidence (84%) and density (mean, 5,314 dpm/mg tissue). Likewise, synovial sarcomas expressed Y1 on tumor cells in high density (mean, 7,497 dpm/mg; incidence, 40%). The remaining tumors expressed NPY receptor subtypes Y1 or Y2 at lower levels. Moreover, many of the sarcomas showed Y1 expression on intratumoral blood vessels. In situ hybridization for Y1 mRNA confirmed the autoradiography results. CONCLUSIONS: NPY receptors are novel molecular markers for human sarcomas. Y1 may inhibit growth of specific sarcomas, as previously shown in an in vivo mouse model of human ESFT. The high Y1 expression on tumor cells of ESFT and synovial sarcomas and on blood vessels in many other sarcomas represents an attractive basis for an in vivo tumor targeting.

Neuropeptide Y receptors in primary human brain tumors: overexpression in high-grade tumors

Peptide receptors are often overexpressed in tumors, and they may be targeted in vivo. We evaluated neuropeptide Y (NPY) receptor expression in 131 primary human brain tumors, including gliomas, embryonal tumors, meningiomas, and pituitary adenomas, by in vitro receptor autoradiography using the 125I-labeled NPY receptor ligand peptide YY in competition with NPY receptor subtype-selective analogs. Receptor functionality was investigated in selected cases using [35S]GTPgammaS-binding autoradiography. World Health Organization Grade IV glioblastomas showed a remarkably high expression of the NPY receptor subtype Y2 with respect to both incidence (83%) and density (mean, 4,886 dpm/mg tissue); astrocytomas World Health Organization Grades I to III and oligodendrogliomas also exhibited high Y2 incidences but low Y2 densities. In glioblastomas, Y2 agonists specifically stimulated [35S]GTPgammaS binding, suggesting that tumoral Y2 receptors were functional. Furthermore, nonneoplastic nerve fibers containing NPY peptide were identified in glioblastomas by immunohistochemistry. Medulloblastomas, primitive neuroectodermal tumors of the CNS, and meningiomas expressed Y1 and Y2 receptor subtypes in moderate incidence and density. In conclusion, Y2 receptors in glioblastomas that are activated by NPY originating from intratumoral nerve fibers might mediate functional effects on the tumor cells. Moreover, identification of the high expression of NPY receptors in high-grade gliomas and embryonal brain tumors provides the basis for in vivo targeting.

Comparative study of the neuropeptide-Y sympathetic nerves in endometriotic involved and noninvolved sacrouterine ligaments in women with pelvic endometriosis

STUDY OBJECTIVE: To show the relationship between the neuropeptide-Y pelvic sympathetic nerves and neoangiogenesis in the development of endometriosis DESIGN: Prospective study. SETTING: Academic community teaching hospital. PATIENTS: Fifteen consecutive women with unilateral endometriotic infiltration of the sacrouterine ligaments. INTERVENTIONS: A laparoscopic excision/biopsy of involved and noninvolved parts of the sacrouterine ligaments were taken. The sections were incubated with the neuronal marker rabbit polyclonal anti-protein gene product 9.5 and rabbit polyclonal anti-neuropeptide-Y. We made a comparative study on the distribution of nerve fibers and their relationship to the vessels on intact and endometriotic involved tissue. MEASUREMENTS AND MAIN RESULTS: The results show that a large amount of nerves are present around the blood vessels in the endometriosis samples, and a large number of these nerves are neuropeptide-Y sympathetic nerves. Adrenergic fibers are also present in the intact control subjects, however, in significantly smaller amounts. CONCLUSION: This finding shows a strong relationship between the neuropeptide-Y sympathetic pelvic nerves and the neoangiogenesis required for the development of endometriosis.

Neuropeptide Y restores non-receptor-mediated vasoconstrictive action in superior mesenteric arteries in portal hypertension

BACKGROUND & AIMS Vascular hyporeactivity to vasoconstrictors contributes to splanchnic arterial vasodilatation and hemodynamic dysregulation in portal hypertension. Neuropeptide Y (NPY), a sympathetic cotransmitter, has been shown to improve adrenergic vascular contractility in portal hypertensive rats and markedly attenuate hyperdynamic circulation. To further characterize the NPY-effects in portal hypertension, we investigated its role for non-receptor-mediated vasoconstriction in the superior mesenteric artery (SMA) of portal vein ligated (PVL) and sham-operated rats. METHODS Ex vivo SMA perfusion of PVL and sham rats was used to analyse the effects of NPY on pressure response to non-receptor-mediated vasoconstriction. Dose-response curves to KCl (30-300 mM) were used to bypass G protein-coupled receptor mechanisms. Potential involvement of the cyclooxygenase-pathway was tested by non-selective cyclooxygenase-inhibition using indomethacin. RESULTS KCl-induced vascular contractility but not vascular sensitivity was significantly attenuated in PVL rats as compared with sham rats. Administration of NPY resulted in an augmentation of KCl-evoked vascular sensitivity being not different between study groups. However, KCl-induced vascular contractility was markedly more enhanced in PVL rats, thus, vascular response was no more significantly different between PVL and sham rats after addition of NPY. Administration of indomethacin abolished the NPY-induced enhancement of vasoconstriction. CONCLUSIONS Receptor-independent vascular contractility is impaired in mesenteric arteries in portal hypertension. NPY improves non-receptor mediated mesenteric vasoconstriction more effective in portal hypertension than in healthy conditions correcting splanchnic vascular hyporesponsiveness. This beneficial vasoactive action of NPY adds to its well known more pronounced effects on adrenergic vasoconstriction in portal hypertension making it a promising therapeutic agent in portal hypertension.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, catecholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

Peptide receptor expression in GEP-NET

Numerous peptide receptors have recently been reported to be expressed or overexpressed in various human cancers. For instance, somatostatin receptors are particularly frequently expressed in gastroenteropancreatic neuroendocrine tumors (GEP-NET), including both primaries and metastases. The density is often high, and the distribution is usually homogenous. While various somatostatin receptor subtypes can be expressed in these tumors, the sst(2) is clearly predominant. These receptors represent the molecular basis for a number of clinical applications, including symptomatic therapy with octreotide in hormone-secreting GEP-NET, in vivo diagnostic with radiolabeled diethylene triamine pentaacetic acid octreotide (Octreoscan) to evaluate the extend of the disease, and (90)Y- or (177)Lu-[(90)Y-DOTA]-D: -Phe(1)-Tyr(3) octreotide radiotherapy. GEP-NET can, however, express peptide receptors other than somatostatin receptor: Insulinomas have more glucagon-like peptide 1 receptors than somatostatin receptors; gastrinomas express very high levels of secretin receptors. GEP-NET may also express cholecystokinin 2, bombesin, neuropeptide Y, or vasoactive intestinal peptide receptors. Often, several of these peptide receptors are expressed simultaneously in GEP-NET, providing a molecular basis for in vivo multireceptor targeting of those tumors.