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The objective of this study was to outline the dynamics of prokineticin-2 pathway in relation to clinical-pathological features of Parkinson's disease by examining olfactory neurons of patients.Thirty-eight patients (26 de novo, newly diagnosed) and 31 sex/age-matched healthy controls underwent noninvasive mucosa brushing for olfactory neurons collection, and standard clinical assessment. Gene expression levels of prokineticin-2, prokineticin-2 receptors type 1 and 2, and prokineticin-2-long peptide were measured in olfactory neurons by real-time polymerase chain reaction (PCR); moreover, the prokineticin-2 protein and α-synuclein species (total and oligomeric) were quantified by immunofluorescence staining.Prokineticin-2 expression was significantly increased in Parkinson's disease. De novo patients had higher prokineticin-2 levels, directly correlated with Movement Disorder Society-Sponsored Revision of the Unified Parkinson Disease Rating Scale (MDS-UPDRS) part III motor score. In addition, oligomeric α-synuclein was higher in Parkinson's disease and directly correlated with prokineticin-2 protein levels. Total α-synuclein did not differ between patients and controls.Prokineticin-2 is a chemokine showing neuroprotective effects in experimental models of Parkinson's disease, but translational proof of its role in patients is still lacking. Here, we used olfactory neurons as the ideal tissue to analyze molecular stages of neurodegeneration in vivo, providing unprecedented evidence that the prokineticin-2 pathway is activated in patients with Parkinson's disease. Specifically, prokineticin-2 expression in olfactory neurons was higher at early disease stages, proportional to motor severity, and associated with oligomeric α-synuclein accumulation. These data, consistently with preclinical findings, support prokineticin-2 as a candidate target in Parkinson's disease, and validate reliability of olfactory neurons to reflect pathological changes of the disease. ANN NEUROL 2022.

Tc85, as other members of trans-sialidase family, is involved in Trypanosoma cruzi parasite adhesion to mammalian cells. Particularly, Tc85 acts through specific interactions with prokineticin receptor 2, a G-protein coupled receptor involved in diverse physiological and pathological processes. In this manuscript, through biochemical analyses, we demonstrated that LamG, a Tc85 domain, physically interacts with the prokineticin receptor 2. Moreover, expressing prokineticin receptor 1 and 2 we demonstrated that LamG specifically activates prokineticin receptor 2 through a strong coupling with Gαi or Gαq proteins in yeast strains and inducing ERK and NFAT phosphorylation in CHO mammalian cells. To demonstrate a Tc85 physiological role in T. cruzi infection of the nervous system, we evidenced a strong STAT3 and ERK activation by LamG in mice Dorsal Root Ganglia. L173R is the most common prokineticin receptor 2 mutation reported in Kallmann syndrome and it is a founder mutation. Our results demonstrated that in cells co-expressing prokineticin receptor 2 mutant (L173R) and wild-type, LamG is unable to induce signal transduction. The L173R mutation in heterozygosity may allow for a selective advantage due to increased protection from T. cruzi infection. SIGNIFICANCE OF THE STUDY: The Chagas' disease affecting millions of people worldwide is caused by an eukaryotic microorganism called T. cruzi. Pharmacological treatment for patients with Chagas' disease is still limited. Indeed, the small number of drugs available shows important side effects that can be debilitating for patient health. In order to replicate and produce new parasites T. cruzi uses a complex of different proteins produced by both the parasite and the human host cells. So, understanding the molecular details used by T. cruzi to be internalised by different types of human cells is an important step towards the development of new drugs for this disease. Prokineticin receptors are relevant for host-parasite interaction. To characterise the signal transduction cascade induced by their activation may help to understand the molecular details of cell infection, leading to novel therapeutic alternative for this debilitating disease.

Background and Purpose Chemokines are involved in neuroinflammation and contribute to chronic pain processing. The new chemokine prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. In the present study, we investigated the involvement of PROK2 and its receptors in neuropathic pain. Experimental Approach Effects of single, intrathecal, perineural and s.c. injections of the PKR antagonist PC1, or of 1 week s.c. treatment, on thermal hyperalgesia and tactile allodynia was evaluated in mice with chronic constriction of the sciatic nerve (CCI). Expression and localization of PROK2 and of its receptors at peripheral and central level was evaluated 10 days after CCI, following treatment for 1 week with saline or PC1. IL-1β and IL-10 levels, along with glia activation, were evaluated. Key Results Subcutaneous, intrathecal and perineural PC1 acutely abolished the CCI-induced hyperalgesia and allodynia. At 10 days after CCI, PROK2 and its receptor PKR2 were up-regulated in nociceptors, in Schwann cells and in activated astrocytes of the spinal cord. Therapeutic treatment with PC1 (s.c., 1 week) alleviated established thermal hyperalgesia and allodynia, reduced the injury-induced overexpression of PROK2, significantly blunted nerve injury-induced microgliosis and astrocyte activation in the spinal cord and restored the physiological levels of proinflammatory and anti-inflammatory cytokines in periphery and in spinal cord. Conclusion and Implications The prokineticin system contributes to pain modulation via neuron–glia interaction. Sustained inhibition of the prokineticin system, at peripheral or central levels, blocked both pain symptoms and some events underlying disease progression.

Background and Purpose Prokineticin 2 (PK2) has recently been shown to acutely reduce food intake in rodents. We aimed to determine the CNS sites and receptors that mediate the anorectic effects of peripherally administered PK2 and its chronic effects on glucose and energy homeostasis. Experimental Approach We investigated neuronal activation following i.p. administration of PK2 using c-Fos-like immunoreactivity (CFL-IR). The anorectic effect of PK2 was examined in mice with targeted deletion of either prokineticin receptor 1 (PKR1) or prokineticin receptor 2 (PKR2), and in wild-type mice following administration of the PKR1 antagonist, PC1. The effect of IP PK2 administration on glucose homeostasis was investigated. Finally, the effect of long-term administration of PK2 on glucose and energy homeostasis in diet-induced obese (DIO) mice was determined. Key Results I.p. PK2 administration significantly increased CFL-IR in the dorsal motor vagal nucleus of the brainstem. The anorectic effect of PK2 was maintained in mice lacking the PKR2 but abolished in mice lacking PKR1 and in wild-type mice pre-treated with PC1. DIO mice treated chronically with PK2 had no changes in glucose levels but significantly reduced food intake and body weight compared to controls. Conclusions and Implications Together, our data suggest that the anorectic effects of peripherally administered PK2 are mediated via the brainstem and this effect requires PKR1 but not PKR2 signalling. Chronic administration of PK2 reduces food intake and body weight in a mouse model of human obesity, suggesting that PKR1-selective agonists have potential to be novel therapeutics for the treatment of obesity.

BACKGROUND AND PURPOSE The amphibian peptide Bv8 induces potent nociceptive sensitization in rodents. Its mammalian homologue, prokineticin 2 (PROK2), is strongly up-regulated in inflamed tissues and is a major determinant in triggering inflammatory pain. Bv8 and PROK2 activate two closely related GPCRs, PK1 and PK2, in a relatively non-selective fashion. To characterize better the roles of the two receptors in hyperalgesia and to obtain ligands whose binding affinity and efficacy differed for the two receptors, we modified the Bv8 molecule in regions essential for receptor recognition and activation. EXPERIMENTAL APPROACH We modified the Bv8 molecule by substituting Trp in position 24 with Ala (A-24) and compared it with Bv8 for binding and activating PK1 and PK2 receptors in cell preparations and in affecting nociceptive thresholds in rodents. KEY RESULTS A-24 preferentially bound to PK2 receptors and activated them with a lower potency (5-fold) than Bv8. When systemically injected, A-24 induced Bv8-like hyperalgesia in rats and in mice, at doses 100 times higher than Bv8. Locally and systemically injected at inactive doses, A-24 antagonized Bv8-induced hyperalgesia. In rat and mouse models of inflammatory and post-surgical pain, A-24 showed potent and long-lasting anti-hyperalgesic activity. Unlike Bv8, A-24 increased β-endorphin levels in mouse brain. CONCLUSIONS AND IMPLICATIONS A-24 induced its anti-hyperalgesic effect in rodents by directly blocking nociceptor PK1 receptors and by activating the central opioid system and the descending pain control pathway through brain PK2 receptors.

The prokineticin Bv8, a small protein secreted by the skin of the Bombina variegata frog, is a potent agonist of both the identified prokineticin receptors, the G-protein-coupled PK-R1 and PK-R2. We found in this study that intraperiaqueductal grey (PAG) Bv8, 100 and 200 pmol per rat, exerted a pronociceptive action and caused opposite effects on the ongoing rostral ventromedial medulla (RVM) On- and Off-cell activities in rats. Bv8 increased and decreased the ongoing activity of RVM On and Off cells, respectively. Bv8 decreased tail flick latency and increased the pause and shortened the onset of the Off-cell pause. Bv8 did not change either the tail flick-induced On-cell burst of activity or the onset of On-cell peak firing. Microdialysis analysis, applied in combination with the plantar test, showed that intra-PAG perfusion with Bv8 (0.25 and 0.5 pm) increased GABA, but not glutamate, extracellular levels, and decreased thermoceptive thresholds. These findings show that stimulation of PAG PK-Rs might worsen pain perception and this effect is consistent with both RVM On- and Off-cell ongoing and tail flick-related activities, as well as with the increase induced by Bv8 in PAG GABA levels.

The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N-terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C-terminal domain. Two closely related G protein-coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK-R1 and PK-R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des-Ala-Bv8) or two (des-Ala-Val-Bv8) residues from the N-terminus. des-Ala-Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca2+]i transients and in causing p42/p44 MAPK phosphorylation in CHO-cells expressing PK-R1 and PK-R2. Moreover, dA-Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. The deletion of the first two amino acids of Bv8 abolished any biological activity both ‘in vitro’ and ‘in vivo’; however, des-AlaVal-Bv8 is able to antagonize the Bv8-induced hyperalgesia, binding the PK-Rs on peripheral and central projections of the primary sensitive neurons. British Journal of Pharmacology (2005) 146, 625–632. doi:10.1038/sj.bjp.0706376

1. The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose mammalian orthologues have been identified and named prokineticins (PK-1 and PK-2). 2. Bv8 (from 2.5 to 60 pmol) injected into the lateral ventricles of rat brain suppressed diurnal, nocturnal, deprivation-induced and neuropeptide Y-stimulated feeding and stimulated diurnal drinking. Nocturnal drinking was increased only in fasted rats. 3. PK-2 mRNA is expressed in discrete areas of the rat brain, including the suprachiasmatic nucleus (SCN), medial preoptic area (MPA) and nucleus of the solitary tract (NTS). In the SCN neurons, PK-2 mRNA is highest during the light phase of the circadian cycle and undetectable during the dark phase. 4. The G-protein-coupled receptor prokineticin receptor 2 (PKR-2), which binds Bv8 and PK-2 with high affinity, is mainly expressed in the piriform cortex, paraventricular thalamic nucleus, parataenial nucleus (PT), SCN, hypothalamic paraventricular (PVH) and dorsomedial (DMH) nuclei, arcuate nucleus (ARC) and subfornical organ (SFO) of the rat brain. 5. Bv8 microinjected into the ARC, at doses from 0.02 to 2.0 pmol during night-time or from 0.2 to 5 pmol in 24-h-fasted rats, selectively suppressed feeding without affecting drinking. When injected into the SFO, Bv8 (from 0.2 to 2 pmol) stimulated drinking but did not affect feeding. Bv8 injections into other brain areas left rat ingestive behaviours unchanged. 6. We hypothesize that PK-2-rich projections from SCN neurons to PKR-expressing ARC neurons could transmit the circadian rhythm of feeding, whereas inputs from the PK-2-expressing NTS neurons to the PKR-2-expressing SFO neurons could transmit visceral information on the water-electrolyte balance and osmotic regulation.

The small protein Bv8, isolated from amphibian skin, belongs to a novel family of secretory proteins (Bv8-Prokineticin family, SWISS-PROT: Q9PW66) whose orthologues have been conserved throughout evolution, from invertebrates to humans. When injected intravenously or subcutaneously (from 0.06 to 500 pmol kg−1) or intrathecally (from 6 fmol to 250 pmol) in rats, Bv8 produced an intense systemic nociceptive sensitization to mechanical and thermal stimuli applied to the tail and paws. Topically delivered into one rat paw, 50 fmol of Bv8 decreased by 50% the nociceptive threshold to pressure in the injected paw without affecting the threshold in the contralateral paw. The two G-protein coupled prokineticin receptors, PK-R1 and PK-R2, were expressed in rat dorsal root ganglia (DRG) and in dorsal quadrants of spinal cord (DSC) and bound Bv8 and the mammalian orthologue, EG-VEGF, with high affinity. In DSC, PK-R1 was more abundant than PK-R2, whereas both receptors were equally expressed in DRG. IC50 of Bv8 and EG-VEGF to inhibit [125I]-Bv8 binding to rat DRG and DSC were 4.1±0.4 nM Bv8 and 76.4±7.6 nM EG-VEGF, in DRG; 7.3±0.9 nM Bv8 and 330±41 nM EG-VEGF, in DSC. In the small diameter neurons (

A new improved synthetic methodology to form triazinediones as potential antagonists for prokineticin receptors is described.

When administered subcutaneously HS-599, a new didehydroderivative of buprenorphine (18,19-dehydrobuprenorphine), produced a long-lasting antinociceptive response in rats. Its potency exceeded twice that of buprenorphine. In the tail-flick test it acted as a full agonist but in the plantar test only as a partial agonist. Whereas the μ-opioid antagonists naloxone and naltrexone antagonized HS-599 antinociception the δ-opioid antagonist naltrindole and the κ-opioid antagonist nor-binaltorphimine did not. Unlike buprenorphine and morphine, HS-599 never induced conditioned place-preference in rats. In radioligand binding assays, compared with buprenorphine HS-599 had 3 fold higher μ-opioid receptor affinity but lower δ- and κ-opioid receptor affinity. In isolated guinea-pig ileum preparations, HS-599 only partially inhibited the electrically-stimulated contraction, acting as a partial opioid agonist. When tested against the μ-opioid receptor agonist dermorphin, it behaved as a non-equilibrium antagonist. Conversely, in mouse vas deferens (rich in δ-opioid receptors) and rabbit vas deferens preparations (rich in κ-opioid receptors) HS-599 acted as a pure equilibrium antagonist, shifting the log-concentration-response curves of the δ-opioid agonist deltorphin I and the κ-opioid agonist U-69593 to the right. In conclusion, HS-599 is a novel buprenorphine derivative with higher affinity, selectivity and potency than the parent compound, for μ-opioid receptors. It produces intense and long-lasting antinociception and does not induce place-preference in rats. British Journal of Pharmacology (2001) 134, 441–447; doi:10.1038/sj.bjp.0704280

To reduce the density of δ-opioid receptor protein, five antisense phosphorothioate oligodeoxynucleotides (aODN), targeting the three exons of rat δ-opioid receptor mRNA (DOR), were injected twice daily for 4 days or continuously infused for 7 days into brain lateral ventricles (i.c.v.) of Sprague-Dawley rats. Rats acting as controls were infused or injected with a mismatch sequence (mODN) of each aODN. The density of opioid receptors in rat brain membranes was measured by saturation binding experiments using selective ligands for δ, μ and κ opioid receptors. aODNs injected twice a day for 4 days left rat brain δ-opioid receptor density unchanged. The ODN targeting the DOR nucleotide sequence 280–299 (aODN280–299, exon 2), decreased brain δ-opioid receptor density significantly more than aODNs targeting exon 1 (aODN239–258), exon 2 (aODN361–380), or exon 3 (aODN741–760) (to 52% vs 79, 72, and 68%). None of the aODNs to the DOR changed the brain density of μ- or k-opioid receptors. When in a novel environment (but not when kept in their home cages), the locomotor activity of aODN280–299 treated rats was significantly lower than that of saline or mODN treated rats. The δ-opioid agonist SNC80 (5 mg kg−1, s.c.) significantly and potently stimulated locomotion and delayed colonic propulsion in saline- and mODN-infused rats, but left motor behaviour and colonic transit of δ-knockdown rats unchanged. The baseline nociceptive threshold and the antinociceptive response to morphine were unchanged in δ-knockdown rats. British Journal of Pharmacology (1999) 128, 1554–1560; doi:10.1038/sj.bjp.0702931

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys7]dermorphin and its main metabolites, [1-5]dermorphin and [1-4]dermorphin. Fifteen minutes after injection, doses of [Lys7]dermorphin producing antinociception (i.c.v., 36–120 nmol; s.c., 0.12–4.7 μmol kg−1) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5-HT receptor antagonist ritanserin (2 mg kg−1, s.c.), was blocked by naloxone (0.1 mg kg−1, s.c.), significantly reduced by the μ1 opioid receptor antagonist naloxonazine (10 mg kg−1, s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg−1, s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3–14.2 μmol kg−1, i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO2. I.c.v. administration of [1-5]dermorphin and [1-4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO2-stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone. In awake rats, [Lys7]dermorphin (0.1–1 mg kg−1, s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine. In conclusion, the present study indicates that analgesic doses of [Lys7]dermorphin stimulate respiration by activating central μ1 opioid receptors and this respiratory stimulation involves a forebrain 5-hydroxytryptaminergic excitatory pathway.

The density and affinity of binding sites for the δ-selective opioid ligands [3H]-[d-Ala2, Asp4]deltorphin (DELT-I), [3H]-[d-Ala2Glu4]-deltorphin (DELT-II), [3H]-[d-Pen2,d-Pen5]enkephalin (DPDPE), and [3H]-naltrindole (NTI) were determined in whole brain from 10, 15, 25 and 60 day-old C57BL mice. At all ages, the analyses of the homologous displacement curves, gave best fits to single rather than to multiple site models. The binding capacity (Bmax) labelled by [3H]-NTI was about one half that labelled by [3H]-DELT-I, [3H]-DELT-II and [3H]-DPDPE. In 25 and 60 day-old mouse brain the DPDPE Bmax was 25% less than the deltorphin-II Bmax. In saturation experiments, specific binding of [3H]-DELT-I on adult mouse brain homogenates was best fitted by a two-site model (34% high affinity site, Kd=1.08 nm and 66% low affinity sites, Kd=39.9 nm). DPDPE produced a biphasic inhibition of specific [3H]-DELTI-I binding, from 15 days of age onwards. The relative percentage of high and low affinity sites was 72% and 28% in 15 day-, 65% and 35% in 25 day- and 30% and 70% in 60 day-old mice. In adult mouse brain labelled with [3H]-DELT-I, DELT-II recognized 71% of high-affinity and 29% of low-affinity sites. DELT-I and DPDPE produced monophasic inhibition of specific [3H]-DELT-II binding to brain homogenates of adult mice. These data suggest that a sub-population of δ-sites (probably the δ2-subtype), recognized by DELT-I, with high affinity for DELT-II and low affinity for DPDPE develops from 25 days onward. In electrically stimulated mouse vas deferens (MVD) the rank order of potency of the three δ-agonists was: DELT-I> DELT-II> DPDPE in 10 day-old mice, and: DELT-I= DELT-II> DPDPE, from 25 days onward. During this time, the potency of DELT-II increased about 15 fold whereas the potency of DELT-I and DPDPE increased only 5 times. The higher efficacy of DELT-II could depend on receptor maturation towards the δ2-subtype. British Journal of Pharmacology (1997) 120, 989–994; doi:10.1038/sj.bjp.0700990