Neuropeptide FF Receptors Research Articles

Hederagenin is a Highly Selective Antagonist of the Neuropeptide FF Receptor 1 that Reveals Mechanisms for Subtype Selectivity

AbstractRF‐amide peptide receptors including the neuropeptide FF receptor 1 (NPFFR1) are G protein‐coupled receptors (GPCRs) that modulate diverse physiological functions. High conservation of endogenous ligands and receptors makes the identification of selective ligands challenging. Previously identified antagonists mimic the C‐terminus of peptide ligands and lack selectivity towards the closely related neuropeptide FF receptor 2 (NPFFR2) or the neuropeptide Y1 receptor (Y1R). In a high‐throughput screening, we identified the pentacyclic triterpenoid hederagenin (1) as a novel selective antagonist for the NPFFR1. Hederagenin (1) is a natural product isolated from Hedera helix (ivy). We characterized its mode of activity using in vitro and in silico methods, revealing an overlapping binding site of the small molecule with the orthosteric peptide agonists. Despite the high similarity of the orthosteric binding pockets of NPFFR1 and NPFFR2, hederagenin (1) shows strong subtype selectivity, particularly caused by slight differences in the shape of the binding pockets and the rigidity of the small molecule. Several residues inhibiting the activity of hederagenin (1) at the NPFFR2 were identified. As NPFFR1 antagonists are discussed as potential candidates for the treatment of chronic pain, these insights into the structural determinants governing subtype specificity will facilitate the development of next‐generation analgesics with improved safety and efficacy.

Hederagenin is a Highly Selective Antagonist of the Neuropeptide FF Receptor 1 that Reveals Mechanisms for Subtype Selectivity.

RF-amide peptide receptors including the neuropeptide FF receptor 1 (NPFFR1) are G protein-coupled receptors (GPCRs) that modulate diverse physiological functions. High conservation of endogenous ligands and receptors makes the identification of selective ligands challenging. Previously identified antagonists mimic the C-terminus of peptide ligands and lack selectivity towards the closely related neuropeptide FF receptor 2 (NPFFR2) or the neuropeptide Y1 receptor (Y1R). In a high-throughput screening, we identified the pentacyclic triterpenoid hederagenin (1) as a novel selective antagonist for the NPFFR1. Hederagenin (1) is a natural product isolated from Hedera helix (ivy). We characterized its mode of activity using in vitro and in silico methods, revealing an overlapping binding site of the small molecule with the orthosteric peptide agonists. Despite the high similarity of the orthosteric binding pockets of NPFFR1 and NPFFR2, hederagenin (1) shows strong subtype selectivity, particularly caused by slight differences in the shape of the binding pockets and the rigidity of the small molecule. Several residues inhibiting the activity of hederagenin (1) at the NPFFR2 were identified. As NPFFR1 antagonists are discussed as potential candidates for the treatment of chronic pain, these insights into the structural determinants governing subtype specificity will facilitate the development of next-generation analgesics with improved safety and efficacy.

The dual modulating effects of neuropeptide FF on morphine-induced analgesia at the spinal level.

Increasing evidence indicates that neuropeptide FF (NPFF) produces analgesic effects and augments opioid-induced analgesia at the spinal level. However, our recent research demonstrated that NPFF exerted complex opioid-modulating effects in an inflammatory pain model after intrathecal (i.t.) injection. Consistent with previous findings, we found that i.t. NPFF dose-dependently attenuated complete Freund's adjuvant-induced pain hypersensitivity. Interestingly, pharmacological results illustrated that NPFF exhibited opposite opioid-modulating effects at the spinal level depending on its administration dosage, wherein i.t. NPFF potentiated morphine-induced anti-allodynia at the dose of 10 nmol, while attenuated morphine analgesia at an ultra-low-dose of 10 pmol. Behavioral results obtained from neuropeptide FF receptor 2 (NPFFR2) knockout animals suggested that both pro- and anti-opioid effects of NPFF were mediated by NPFFR2. Moreover, these modulating effects of spinal NPFFR2 were selectively targeting mu-opioid receptor, had no effect on delta- and kappa-opioid receptor agonist-induced analgesia. Finally, the opioid-modulating effects of NPFF were further verified using in vitro calcium imaging assay, demonstrating that pretreated with NPFF in primary-cultured spinal neurons significantly attenuated the inhibitory effects of morphine on high-K+-induced neuronal excitability. Taken together, our results suggested that NPFF exhibited dual modulating effects on morphine-induced analgesia after i.t. administration, which provides a possible mechanism to explain the complex opioid-modulating effects of endogenous NPFF systems.

Neuropeptide FF prevented histamine- or chloroquine-induced acute itch behavior through non-NPFF receptors mechanism in male mice

The neuropeptide FF (NPFF) system regulates various physiological and pharmacological functions, particularly pain modulation. However, the modulatory effect of NPFF system on itch remains unclear. To investigate the modulatory effect and functional mechanism induced by NPFF system on acute itch, we examined the effects of supraspinal administration of NPFF and related peptides on acute itch induced by intradermal (i.d.) injection of histamine or chloroquine in male mice. Our results indicated that intracerebroventricular (i.c.v.) administration of NPFF dose-dependently prevented histamine- or chloroquine-induced acute itch behaviors. In addition, the modulatory effect of NPFF was not affected by the selective NPFF receptor antagonist RF9. Furthermore, we investigated the effects of NPVF and dNPA, the selective agonists of NPFF1 and NPFF2 receptors respectively, on the acute itch. The results demonstrated that both NPFF agonists effectively prevented acute itch induced by histamine or chloroquine in a manner similar to NPFF, and their effects were also not modified by RF9. To further investigate the possible mechanism of the NPFF receptors agonists, the NPFF-derived analogues [Phg8]-NPFF and NPFF(1–7)-NH2 that could not activate NPFF receptors in cAMP assays were subsequently tested in the acute itch model. Interestingly, [Phg8]-NPFF, but not NPFF(1–7)-NH2, prevented acute itch behavior after i.c.v. administration. In conclusion, our findings reveal that NPFF and related peptides prevent histamine- and chloroquine-induced acute itch through a NPFF receptor-independent mechanism. And it was revealed that the C-terminal phenyl structure of NPFF may play a crucial role in these modulatory effects on acute itch.

Deficiency of GPR10 and NPFFR2 receptors leads to sex-specific prediabetic syndrome and late-onset obesity in mice.

GPR10 and neuropeptide FF receptor 2 (NPFFR2) play important role in the regulation of food intake and energy homeostasis. Understanding the interaction between these receptors and their specific ligands, such as prolactin-releasing peptide, is essential for developing stable peptide analogs with potential for treating obesity. By breeding and characterizing double knockout (dKO) mice fed standard or high-fat diet (HFD), we provide insights into the metabolic regulation associated with the GPR10 and NPFFR2 deficiency. Both WT and dKO mice were subjected to behavioral tests and an oral glucose tolerance test. Moreover, dual-energy X-ray absorptiometry (DEXA) followed by indirect calorimetry were performed to characterize dKO mice. dKO mice of both sexes, when exposed to an HFD, showed reduced glucose tolerance, hyperinsulinemia, and insulin resistance compared with controls. Moreover, they displayed increased liver weight with worsened hepatic steatosis. Mice displayed significantly increased body weight, which was more pronounced in dKO males and caused by higher caloric intake on a standard diet, while dKO females displayed obesity characterized by increased white adipose tissue and enhanced hepatic lipid accumulation on an HFD. Moreover, dKO females exhibited anxiety-like behavior in the open field test. dKO mice on a standard diet had a lower respiratory quotient, with no significant changes in energy expenditure. These results provide insights into alterations associated with disrupted GPR10 and NPFFR2 signaling, contributing to the development of potential anti-obesity treatment.

Intrathecal administration of MCRT produced potent antinociception in chronic inflammatory pain models via μ-δ heterodimer with limited side effects

An important goal in the opioid field is to discover effective analgesic drugs with minimal side effects. MCRT demonstrated potent antinociceptive effects with limited side effects, making it a promising candidate. However, its pharmacological properties and how it minimizes side effects remain unknown. Various mouse pain and opioid side effect models were used to evaluate the antinociceptive properties and safety at the spinal level. The targets of MCRT were identified through cAMP measurement, isolated tissue assays, and pharmacological experiments. Immunofluorescence was employed to visualize protein expression. MCRT displayed distinct antinociceptive effects between acute and chronic inflammatory pain models due to its multifunctional properties at the μ opioid receptor (MOR), µ-δ heterodimer (MDOR), and neuropeptide FF receptor 2 (NPFFR2). Activation of NPFFR2 reduced MOR-mediated antinociception, leading to bell-shaped response curves in acute pain models. However, activation of MDOR produced more effective antinociception in chronic inflammatory pain models. MCRT showed limited tolerance and opioid-induced hyperalgesia in both acute and chronic pain models and did not develop cross-tolerance to morphine. Additionally, MCRT did not exhibit addictive properties, gastrointestinal inhibition, and effects on motor coordination. Mechanistically, peripheral chronic inflammation or repeated administration of morphine and MCRT induced an increase in MDOR in the spinal cord. Chronic administration of MCRT had no apparent effect on microglial activation in the spinal cord. These findings suggest that MCRT is a versatile compound that provides potent antinociception with minimal opioid-related side effects. MDOR could be a promising target for managing chronic inflammatory pain and addressing the opioid crisis.

Abstract P422: Neuropeptide FF and Its Receptor NPFFR2 Decrease Renal Sodium Excretion and Increase Blood Pressure in Mice

The kidney is critical in the overall regulation of fluid and electrolyte balance and blood pressure. Neuropeptide FF (NPFF), a morphine-modulating peptide, regulates cardiovascular function through its interaction with two receptors, NPFFR1 and NPFFR2. We now report that NPFF and its receptors, mainly NPFFR2, are expressed in the kidney. NPFF (9.25 mmol, 0.5 mL/hr, 0.05 nmol/day, 7 days) chronically infused underneath the renal capsule, decreased renal sodium excretion (UNaV) from 0.68±0.07 mEq/day, n=5 to 0.43±0.06 mEq/day (n=6/group) in conscious C57BL/6 mice. This was accompanied by an increase in systolic blood pressure (SBP, 114.5±5.0 mm Hg, n=4); the infusion of vehicle (saline) did not affect the blood pressure (96.4±3.0 mm Hg, n=4). The renal subcapsular injection of a single dose (10 µg in 100 µL) of NPFF also increased SBP within 15-35 min (NPFF: 105.5±3.44 mm Hg, n=6); saline injection did not affect SBP (82.6±5.65 mm Hg, n=4). RF-9 (10 µg in 100 µL), an antagonist of NPFF receptors prevented the NPFF-mediated increase in SBP (NPFF alone: 105.5±3.44 mm Hg, n=6; RF+NPFF: 88.5±5.90 mm Hg, n=5), whereas RF-9, alone, had no effect (Baseline: 97.6±2.70 mmHg; RF-9: 90.9±4.70 mm Hg, n=5). The renal subcapsular injection of scrambled peptide (10 µg in 100 µL) had no effect on SBP (86.1±1.83 mm Hg, n=4). However, SBP was decreased by the renal subcapsular infusion of Npffr2 siRNA (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) in C57Bl/6 mice fed with a high salt diet (4% NaCl). Furthermore, the SBP of conventional germline Npffr2 knockout (KO) mice fed a high salt (4% NaCl) diet was lower than the SBP of wild-type (WT) littermates (WT: 105.6±2.9 mm Hg, n=5; KO: 90.5±5.5 mm Hg, n=4, P < 0.05). By contrast, in mice fed normal salt (0.8% NaCl) diet, SBP was not different between Npffr2 KO mice and WT littermates (WT: 96.5±4.5 mm Hg, n=5; KO: 98.7±11.7 mm Hg, n=4, P > 0.05). Taken together, NPFF and its receptor, NPFFR2, in the kidney cause salt-sensitive hypertension in mice. Uncovering the functional relevance of renal NPFF in the dynamic regulation of renal sodium transport will lead to a better understanding of blood pressure homeostasis.

Abstract Mo138: Renal NPFFR2-mediated increase in blood pressure is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule

Neuropeptide FF (NPFF), an octapeptide originally found in the brain, participates in the regulation of blood pressure because it is present with its receptors, NPFFR1 and NPFFR2, in the cardiovascular regulatory center in the hypothalamus. However, the role of NPFF and its receptors in the kidney on blood pressure regulation is not known. In both the human and mouse renal proximal tubule, NPFFR2, rather than NPFFR1, is the predominant NPFF receptor determined by RNA in situ hybridization and immunofluorescence microscopy. In mouse renal proximal tubule cells, AC263093 (10 -6 M, 15 min), a specific NPFFR2 agonist and NPFFR1 antagonist, inhibited the forskolin-stimulated cAMP production (Vehicle: 100±6.14%, n=6; AC263093: 72.4±6.53%, n=6, p<0.05), which was reversed by pretreatment with RF-9, an antagonist of both NPFFR1 and NPFFR2 (98.2±9.10%, n=6); RF-9, by itself, had no significant effect (100.8±11.44%, n=6), indicating linkage of NPFFR2 to the inhibitory G protein Gai in renal proximal tubule cells. In human renal proximal tubule cells, NPFF also increased Na + /K + -ATPase protein expression, determined by immunoblotting, in a time- and concentration-dependent manner. Protein expression of Na + /K + -ATPase was decreased in NPFFR2 -deficient human renal proximal tubule cells caused by specific NPFFR2 siRNA (Mock: 100±1.71%, NPFFR2 siRNA: 58.4±4.93%, n=4; P < 0.05). Furthermore, blood pressure, measured by telemetry, was increased and sodium excretion was decreased in conscious C57Bl/6 mice infused with NPFF (9.25 mmol, 0.5 mL/hr/7 days) underneath the renal capsule. The effect was probably via NPFFR2 because the blood pressure of mice was increased by the intraperitoneal injection of AC263093 (20 mg/kg/day, 7 days) and fed a high salt diet (4% NaCl), which is consistent with the decrease in blood pressure (98±4 vs 113±3 mmHg; P < 0.05; n=3-5/group) by the renal subcapsular infusion of Npffr2 siRNA in C57Bl/6 mice also fed the high salt diet. Taken together, NPFFR2 activation increases blood pressure and decreases sodium excretion that is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule.

Renal autocrine neuropeptide FF (NPFF) signaling regulates blood pressure

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.

GPCR–MAPK signaling pathways underpin fitness trade-offs in whitefly

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Brain RFamide Neuropeptides in Stress-Related Psychopathologies.

The RFamide peptide family is a group of proteins that share a common C-terminal arginine-phenylalanine-amide motif. To date, the family comprises five groups in mammals: neuropeptide FF, LPXRFamides/RFamide-related peptides, prolactin releasing peptide, QRFP, and kisspeptins. Different RFamide peptides have their own cognate receptors and are produced by different cell populations, although they all can also bind to neuropeptide FF receptors with different affinities. RFamide peptides function in the brain as neuropeptides regulating key aspects of homeostasis such as energy balance, reproduction, and cardiovascular function. Furthermore, they are involved in the organization of the stress response including modulation of pain. Considering the interaction between stress and various parameters of homeostasis, the role of RFamide peptides may be critical in the development of stress-related neuropathologies. This review will therefore focus on the role of RFamide peptides as possible key hubs in stress and stress-related psychopathologies. The neurotransmitter coexpression profile of RFamide-producing cells is also discussed, highlighting its potential functional significance. The development of novel pharmaceutical agents for the treatment of stress-related disorders is an ongoing need. Thus, the importance of RFamide research is underlined by the emergence of peptidergic and G-protein coupled receptor-based therapeutic targets in the pharmaceutical industry.

Multitarget μ-Opioid Receptor Agonists─Neuropeptide FF Receptor Antagonists Induce Potent Antinociception with Reduced Adverse Side Effects.

The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.

Guanidine-to-piperidine switch affords high affinity small molecule NPFF ligands with preference for NPFF1-R and NPFF2-R subtypes

The Neuropeptide FF (NPFF) receptor system is known to modulate opioid actions and has been shown to mediate opioid-induced hyperalgesia and tolerance. The lack of subtype selective small molecule compounds has hampered further exploration of the pharmacology of this receptor system. The vast majority of available NPFF ligands possess a highly basic guanidine group, including our lead small molecule, MES304. Despite providing strong receptor binding, the guanidine group presents a potential pharmacokinetic liability for in vivo pharmacological tool development. Through structure-activity relationship exploration, we were able to modify our lead molecule MES304 to arrive at guanidine-free NPFF ligands. The novel piperidine analogues 8b and 16a are among the few non-guanidine based NPFF ligands known in literature. Both compounds displayed nanomolar NPFF-R binding affinity approaching that of the parent molecule. Moreover, while MES304 was non-subtype selective, these two analogues presented new starting points for subtype selective scaffolds, whereby 8b displayed a 15-fold preference for NPFF1-R, and 16a demonstrated an 8-fold preference for NPFF2-R. Both analogues showed no agonist activity on either receptor subtype in the in vitro functional activity assay, while 8b displayed antagonistic properties at NPFF1-R. The calculated physicochemical properties of 8b and 16a were also shown to be more favorable for in vivo tool design. These results indicate the possibility of developing potent, subtype selective NPFF ligands devoid of a guanidine functionality.

Peripheral administration of lipidized NPAF and NPFF analogs does not influence central food intake regulation but induces anxiety-like behavior

RF-amide peptides influence multiple physiological processes, including the regulation of appetite, stress responses, behavior, and reproductive and endocrine functions. In this study, we examined the roles of neuropeptide FF receptors (NPFFR1 and NPFFR2) by generating several lipidized analogs of neuropeptide AF (NPAF) and 1DMe, a stable analog of neuropeptide FF (NPFF). These analogs were administered peripherally for the first time to investigate their effects on food intake and other potential physiological outcomes. Lipidized NPAF and 1DMe analogs exhibited enhanced stability and increased pharmacokinetics. These analogs demonstrated preserved high affinity for NPFFR2 in the nanomolar range, while the binding affinity for NPFFR1 was tens of nanomoles. They activated the ERK and Akt signaling pathways in cells overexpressing the NPFFR1 and NPFFR2 receptors.Acute food intake in fasted mice decreased after the peripheral administration of oct-NPAF or oct-1DMe. However, this effect was not as pronounced as that observed after the injection of palm11-PrRP31, a potent anorexigenic compound used as a comparator that binds to GPR10 and the NPFFR2 receptor with high affinity. Neither oct-1DMe nor oct-NPAF decreased food intake or body weight in mice with diet-induced obesity during long-term treatment. In mice treated with oct-1DMe, we observed decreased activity in the central zone during the open field test and decreased activity in the open arms of the elevated plus maze. Furthermore, we observed a decrease in plasma noradrenaline levels and an increase in plasma corticosterone levels, as well as an increase in Crh expression in the hypothalamus. Moreover, neuronal activity in the hypothalamus was increased after treatment with oct-1DMe.In this study, we report that oct-1DMe did not have any long-term effects on the central regulation of food intake; however, it caused anxiety-like behavior.

The blockade of neuropeptide FF receptor 1 and 2 differentially contributed to the modulating effects on fentanyl-induced analgesia and hyperalgesia in mice

Neuropeptide FF (NPFF) plays a critical role in various physiological processes through the activation of neuropeptide FF receptor 1 and 2 (NPFFR1 and NPFFR2). Numerous evidence has indicated that NPFF exhibits opposite opioid-modulating effects on opioid-induced analgesia after supraspinal and spinal administrations, while the detailed role of NPFFR1 and NPFFR2 remains unclear. In this study, we employed pharmacological and genetic inhibition of NPFFR to investigate the modulating roles of central NPFFR1 and NPFFR2 in opioid-induced analgesia and hyperalgesia, using a male mouse model of acute fentanyl-induced analgesia and secondary hyperalgesia. Our findings revealed that intrathecal (i.t.) injection of the nonselective NPFFR antagonist RF9 significantly enhanced fentanyl-induced analgesia, whereas intracerebroventricular (i.c.v.) injection did not show the same effect. Moreover, NPFFR2 deficient (npffr2−/−) mice exhibited stronger analgesic responses to fentanyl compared to wild type (WT) or NPFFR1 knockout (npffr1−/−) mice. Intrathecal injection of RF9 in npffr1−/− mice also significantly enhanced fentanyl-induced analgesia. These results indicate a crucial role of spinal NPFFR2 in the enhancement of opioid analgesia. Contrastingly, hyperalgesia induced by fentanyl was markedly reversed in npffr1−/− mice but remained unaffected in npffr2−/− mice. Similarly, i.c.v. injection of the selective NPFFR1 antagonist RF3286 effectively prevented fentanyl-induced hyperalgesia in WT or npffr2−/− mice. Notably, co-administration of i.c.v. RF3286 and i.t. RF9 augmented fentanyl-induced analgesia while reducing hyperalgesia. Collectively, these findings highlight the modulating effects of blocking spinal NPFFR2 and supraspinal NPFFR1 on fentanyl-induced analgesia and hyperalgesia, respectively, which shed a light on understanding the pharmacological function of NPFF system in future studies.

The Relationship between Serum Neuropeptide FFR2, Serum Smoothelin and Pregnancy Outcomes in Pregnant Women with Gestational Hypertension

Gestational hypetension is a major public health concern due to its links with cardiovascular disease, stroke and neonatal morbidity and mortality. Timely diagnosis and effective management of gestational hypertension are essential for both maternal and neonatal health. Neuropeptide FF Receptor 2 (NPFFR2) is a protein secreted by the brain and placenta, involved in pain regulation, water balance, and the modulation of cardiovascular effects. This study aims to conduct a comparative analysis of NPFFR2, smoothelin (SMTH), echocardiographic results and pregnancy outcomes in pregnant women with and without gestational hypertension. This study included 78 pregnant participants, which were grouped into women with gestational hypertension (n = 39) and those without gestational hypetension (n = 39). The gestational hypertension population was classified into two groups, i.e., dipper and non-dipper groups, based on the 24-hour ambulatory blood pressure monitoring results. Smoothelin and NPFFR2 analyses were performed using the blood samples and placental tissue samples collected from all participants, along with echocardiography and 24-hour ambulatory blood pressure monitoring. The study group comprised 78 pregnant women with a mean age of 28.8 years and mean gestational age of 27.7 weeks. The gestational hypertension group had a significantly higher NPFFR2 levels, lower SMTH levels and gestational age at birth and higher all 24-hour ambulatory blood pressure monitoring findings. The left atrial-to-aortic ratio and Tricuspid annular plane systolic excursion (TAPSE) were significantly higher in GİH group than in the control group. NPFFR2 and gestational age at birth were found to be independently associated with neonatal intensive care unit admission. Serum NPFFR2 levels were increased in women with gestational hypertension, SMTH levels were decreased, and pregnancy prognosis was found to be associated with NPFFR2 levels.

All-Hydrocarbon Stapled Peptide Multifunctional Agonists at Opioid and Neuropeptide FF Receptors: Highly Potent, Long-Lasting Brain Permeant Analgesics with Diminished Side Effects.

Our previous study reported the multifunctional agonist for opioid and neuropeptide FF receptors DN-9, along with its cyclic peptide analogues c[D-Cys2, Cys5]-DN-9 and c[D-Lys2, Asp5]-DN-9. These analogues demonstrated potent antinociceptive effects with reduced opioid-related side effects. To develop more stable and effective analgesics, we designed, synthesized, and evaluated seven hydrocarbon-stapled cyclic peptides based on DN-9. In vitro calcium mobilization assays revealed that most of the stapled peptides, except 3, displayed multifunctional agonistic activities at opioid and neuropeptide FF receptors. Subcutaneous administration of all stapled peptides resulted in effective and long-lasting antinociceptive activities lasting up to 360 min. Among these stapled peptides, 1a and 1b emerged as the optimized compounds, producing potent central antinociception following subcutaneous, intracerebroventricular, and oral administrations. Additionally, subcutaneous administration of 1a and 1b caused nontolerance antinociception, with limited occurrence of constipation and addiction. Furthermore, 1a was selected as the final optimized compound due to its wider safety window compared to 1b.

Abstract P1014: Renal Autocrine Npff, Via Its Local Receptors, Regulates Blood Pressure Through Its Interaction With The Renal Dopaminergic System

Neuropeptide FF (NPFF), a hormone originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the role of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, is unknown. In this study, we found that NPFF ( NPFF , Npff ), NPFF-R1( NPFFR1 , Npffr1 ), and NPFF-R2( NPFFR2 , Npffr2 ) mRNAs and proteins were expressed in both mouse and human renal proximal tubules (RPTs), determined by RT-PCR, RNAScope, immunoblotting, and immunofluorescence microscopy. NPFF-R2, compared with NPFF-R1, was predominantly expressed in RPTs and RPT cells (RPTCs). In human and mouse RPTCs, NPFF decreased cAMP production in a concentration- and time-dependent manner, consistent with its receptors’ linkage to the inhibitory G protein, Gαi. The D 1 R, but not D 5 R, colocalized with NPFF-R2 in human RPTCs and human kidney tissue sections. The colocalization between D 1 R and NPFF-R2 was decreased by the treatment with NPFF (100 nM, 30 min) in mouse RPTCs. Consistent with these findings, D 1 R co-immunoprecipitated with NPFF-R2 in human RPTCs. Fenoldopam, a D1-like receptor agonist, increased the cellular cAMP production and intracellular Na + accumulation, which were attenuated by NPFF (100 nM, 30 min), indicating an antagonistic interaction between NPFF and D1-like receptors. Moreover, acute and chronic administration of NPFF (10 μg in 100 μL, 0.5 μL/hr) in C57Bl/6 mice increased their blood pressures (from 96.4±3 to 114±5 mmHg, n=4) that were blocked by RF9 (10 μg in 100 μL, 0.5 μL/hr), an antagonist of both NPFF-R1 and NPFF-R2. NPFF also decreased urinary sodium excretion (from 0.36±0.06 to 0.28±0.05 mEq/day, n=4). In summary, NPFF by its negative interaction with the renal dopaminergic system regulates sodium excretion and blood pressure.

Differential activation of neuropeptide FF receptors by gonadotropin-inhibitory hormone peptides in the European sea bass

Neuropeptide FF (NPFF) and gonadotropin-inhibitory hormone (GnIH) are thought to be paralogous, and a recent study has revealed that both NPFF and GnIH peptides can activate the GnIH receptor (GnIHR, also called NPFFR1) in the European sea bass (Dicentrarchus labrax). However, whether GnIH can bind to the NPFF receptor (NPFFR2) is still yet unknown in this species. Accordingly, we further investigated the potential interactions between GnIH and NPFFR2 (two NPFFR2 forms present in sea bass, namely NPFFR2-1 and NPFFR2-2) on the intracellular signaling pathways. Neither GnIH1 nor GnIH2 had any effect on basal CRE-luc activity, while forskolin-stimulated CRE-luc activity was significantly reduced when COS-7 cells expressing sea bass NPFFR2-1 and NPFFR2-2 were challenged with these two GnIH peptides. NPFF and NPAF also inhibited forskolin-induced CRE-luc activity via their cognate receptors. An evident stimulation of SRE-luc activity was observed when COS-7 cells transfected with NPFFR2-1 and NPFFR2-2 were treated with NPFF and NPAF, whereas GnIH peptides had no effect, except a slight but significant increase elicited by 1000 nM of GnIH1 in COS-7 cells expressing NPFFR2-2. Moreover, only GnIH2 exerted an inhibitory action on NFAT-RE-luc activity in COS-7 cells expressing NPFFR2-1. None of GnIH or NPFF peptides altered ERK phosphorylation levels via NPFFR2 receptors. Our results provide new evidence that sea bass GnIH peptides may exert their functions partially via NPFFR2, and PKA, PKC and Ca2+ routes are potential mediators.

RFamide-related Peptide 3 Signaling via Neuropeptide FF Receptor Stimulates Prolactin Secretion in Female Rats.

The RF-amide peptides comprise a family of neuropeptides that includes the kisspeptin (Kp), the natural ligand of kisspeptin receptor (Kiss1r), and the RFamide-related peptide 3 (RFRP-3) that binds preferentially to the neuropeptide FF receptor 1 (Npffr1). Kp stimulates prolactin (PRL) secretion through the inhibition of tuberoinfundibular dopaminergic (TIDA) neurons. Because Kp also has affinity to Npffr1, we investigated the role of Npffr1 in the control of PRL secretion by Kp and RFRP-3. Intracerebroventricular (ICV) injection of Kp increased PRL and LH secretion in ovariectomized, estradiol-treated rats. The unselective Npffr1 antagonist RF9 prevented these responses, whereas the selective antagonist GJ14 altered PRL but not LH levels. The ICV injection of RFRP-3 in ovariectomized, estradiol-treated rats increased PRL secretion, which was associated with a rise in the dopaminergic activity in the median eminence, but had no effect on LH levels. The RFRP-3-induced increase in PRL secretion was prevented by GJ14. Moreover, the estradiol-induced PRL surge in female rats was blunted by GJ14, along with an amplification of the LH surge. Nevertheless, whole-cell patch clamp recordings showed no effect of RFRP-3 on the electrical activity of TIDA neurons in dopamine transporter-Cre recombinase transgenic female mice. We provide evidence that RFRP-3 binds to Npffr1 to stimulate PRL release, which plays a role in the estradiol-induced PRL surge. This effect of RFRP-3 is apparently not mediated by a reduction in the inhibitory tone of TIDA neurons but possibly involves the activation of a hypothalamic PRL-releasing factor.