Mapping the interaction site for ß-arrestin-2 in the prokineticin 2 receptor.

G protein-coupled receptors (GPCRs) are a family of cell membrane receptors that couple and activate heterotrimeric G proteins and their associated intracellular signalling processes after ligand binding. Although the carboxyl terminal of the receptors is essential for this action, it can also serve as a docking site for regulatory proteins such as the ß-arrestins. Prokineticin receptors (PKR1 and PKR2) are a new class of GPCRs that are able to activate different classes of G proteins and form complexes with ß-arrestins after activation by the endogenous agonists PK2. The aim of this work was to define the molecular determinants within PKR2 that are required for ß-arrestin-2 binding and to investigate the role of ß-arrestin-2 in the signalling pathways induced by PKR2 activation. Our data show that PKR2 binds constitutively to ß-arrestin-2 and that this process occurs through the core region of the receptor without being affected by the carboxy-terminal region. Indeed, a PKR2 mutant lacking the carboxy-terminal amino acids retains the ability to bind constitutively to ß-arrestin-2, whereas a mutant lacking the third intracellular loop does not. Overall, our data suggest that the C-terminus of PKR2 is critical for the stability of the ß-arrestin-2-receptor complex in the presence of PK2 ligand. This leads to the ß-arrestin-2 conformational change required to initiate intracellular signalling that ultimately leads to ERK phosphorylation and activation.

The prokineticin Bv8 sensitizes cutaneous terminals of female mice to heat.

BACKGROUND: Injection of the noxious peptide Bv8 has previously been shown to induce a biphasic thermal hyperalgesia in rodents, the first peak presumably due to peripheral sensitization. This hypothesis has never been directly confirmed. We have assessed whether Bv8 can indeed sensitize peripheral nerve fibres in the mouse to heat. METHODS: We used recordings from single cutaneous fibres, cutaneous calcitonin gene-related peptide (CGRP) release and immunostaining in nerves and plantar skin to evaluate the Bv8 effects on cutaneous nerves. RESULTS: Application of Bv8 at nanomolar concentrations (30-310 nmol/L) to skin preparations significantly increased the heat-induced discharge, the heat-induced afterdischarge and reduced threshold temperature of single unmyelinated polymodal fibres. Furthermore, application of Bv8 to hind-paw skin flaps or trigeminal ganglia significantly elevated their heat-induced CGRP release. Capsaicin-induced and to a lesser extent also KCl-induced CGRP releases were also augmented after Bv8 treatment. Immunohistochemistry revealed co-localization of prokineticin 2 (Bv8 ortholog in rodents) and CGRP in both plantar skin and nerve tissues. These results confirm that Bv8 sensitizes cutaneous nerve endings to heat, partly, although not exclusively through TRPV1 activation. CONCLUSION: Our results thus support the hypothesis that the first hyperalgesic phase to follow Bv8 injection to hind paws of intact animals is due to peripheral sensitization of nociceptors. WHAT DOES THIS STUDY ADD?: Our data provide mechanistic insights into the effect Bv8 application exerts on afferent nerve endings and into the concomitant development of thermal hyperalgesia.

PC1, a non-peptide PKR1-preferring antagonist, reduces pain behavior and spinal neuronal sensitization in neuropathic mice.

Peripheral neuropathy is characterized by abnormal pain responses triggered by the release of several mediators and neuronal hyperexcitability at the spinal cord level. Emerging evidence indicates that the enhanced activity of dorsal horn neurons requires communication with glia and microglia, cells that are physiologically involved in neuronal wellbeing. Prokineticins (PKs), which include PK1 and PK2, represent a novel family of chemokines characterized by a unique structural motif comprising five disulfide bonds. They are expressed in the peripheral and central nervous system. PKs bind two G protein coupled receptors, PKR1 and PKR2, and participate in the regulation of several biological processes, including pain sensation. This study aimed to investigate the anti-nociceptive effect of PC1, a non-peptide PKR1-preferring antagonist, in a mouse model of neuropathic pain. To do this, we assessed the activity of spinal cord nociceptive neurons as well as astrocyte and microglia phenotypes after repeated administration of PC1 in vivo. PC1 treatment strongly delayed the development of thermal hyperalgesia and tactile and mechanical allodynia. It also reduced spinal microglial and glial activation 8 days post injury in spared nerve injury (SNI) mice. Neuropathic mice showed an increased level of PK2 protein in the spinal cord, mostly in astrocytes. PC1 treatment completely reversed the increased responsiveness to mechanical stimuli, the decreased threshold of neuronal activation, and the increased spontaneous activity that were observed in nociceptive specific (NS) neurons of SNI mice.

Controlling the activation of the Bv8/prokineticin system reduces neuroinflammation and abolishes thermal and tactile hyperalgesia in neuropathic animals.

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-1ß 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.

Association between outbreaks of highly pathogenic avian influenza subtype H5N1 and migratory waterfowl (family Anatidae) populations.

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 threatens poultry production and human health. Understanding the role that migratory waterfowl play in introducing and maintaining this infection is critical to control the outbreaks. A study was conducted to determine if the occurrence of HPAI subtype H5N1 outbreaks in village poultry in Romania, 2005-2006, was associated with proximity to populations of migratory waterfowl. Reported outbreaks--which could be grouped into three epidemic phases--and migratory waterfowl sites were mapped. The migratory waterfowl site closest to each outbreak was identified. The distances between outbreaks occurring in phase 1 and 2 of the epidemic and the closest migratory waterfowl site were significantly (P<0.001) less than in phase 3, but these distances were only useful in predicting when outbreaks occurred during phase 1 (October-December, 2005) of the epidemic. A spatial lag (rho=0.408, P=0.041) model best fit the data, using distance and [distance]*[distance] as predictors (R2=0.425). The correlation between when outbreaks were predicted to occur and when they were observed to occur was 0.55 (P=0.006). Results support the hypothesis that HPAI virus subtype H5N1 infections of village poultry in Romania during the autumn of 2005 might have occurred via exposure to migratory populations of waterfowl.

Estimation of the basic reproductive number (R0) for epidemic, highly pathogenic avian influenza subtype H5N1 spread.

Three different methods were used for estimating the basic reproductive number (R0) from data on 110 outbreaks of highly pathogenic avian influenza (HPAI) subtype H5N1 that occurred in village poultry in Romania, 12 May to 6 June 2006. We assumed a village-level infectious period of 7 days. The methods applied were GIS-based identification of nearest infectious neighbour (based on either Euclidean or road distance), the method of epidemic doubling time, and a susceptible-infectious (SI) modelling approach. In general, the estimated basic reproductive numbers were consistent: 2.14, 1.95, 2.68 and 2.21, respectively. Although the true basic reproductive number in this epidemic is unknown, results suggest that the use of a range of methods might be useful for characterizing epidemics of infectious diseases. Once the basic reproductive number has been estimated, better control strategies and targeted surveillance programmes can be designed.