Roberta Lattanzi

Impaired Nociception and Inflammatory Pain Sensation in Mice Lacking the Prokineticin Receptor PKR1: Focus on Interaction between PKR1 and the Capsaicin Receptor TRPV1 in Pain Behavior

Bv8, prokineticin-1 or EG-VEGF (endocrine gland-derived vascular endothelial growth factor), and prokineticin-2, are naturally occurring peptide agonists of two G-protein-coupled receptors (GPCRs), prokineticin receptor 1 (PKR1) and PKR2. PKRs are expressed in neurons in the CNS and peripheral nervous system and many dorsal root ganglion (DRG) cells expressing PKRs also express transient receptor potential vanilloid receptor-1 (TRPV1). Mice lacking the pkr1 gene were generated to explore the role of the PKR1 receptor in nociceptive signaling and in nociceptor sensitization. When compared with wild-type littermates, mice lacking the pkr1 gene showed impaired responsiveness to noxious heat, mechanical stimuli, capsaicin, and protons. In wild-type mice, activation of PKRs by the PKR agonist Bv8 caused hyperalgesia and sensitized to the actions of capsaicin. pkr1-null mice exhibited impaired responses to Bv8 but showed normal hyperalgesic responses to bradykinin and PGE2 (prostaglandin E2). Conversely, trpv1-null mice showed a reduced pronociceptive response to Bv8. Additionally, pkr1-null mice showed diminished thermal hyperalgesia after acute inflammation elicited by mustard oil and reduced pain behavior after chronic inflammation produced by complete Freunds adjuvant. The number of neurons that responded with a [Ca2+]i increase to Bv8 exposure was five times lower in pkr1-null DRG cultures than in wild-type cultures. Furthermore, Bv8-responsive neurons from pkr1-null mice showed a significant reduction in the [Ca2+]i response to capsaicin. These findings indicate a modulatory role of PKR1 in acute nociception and inflammatory pain and disclose a pharmacological interaction between PKR1 and TRPV1 in nociceptor activation and sensitization.

Nociceptive sensitization by the secretory protein Bv8

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 (<30 μm) of rat DRG cultures, Bv8 concentrations, ranging from 0.2 to 10 nM, raised [Ca2+]i in a dose‐dependent manner. These data suggest that Bv8, through binding to PK receptors of DSC and primary sensitive neurons, results in intense sensitization of peripheral nociceptors to thermal and mechanical stimuli.

Sensitization of Transient Receptor Potential Vanilloid 1 by the Prokineticin Receptor Agonist Bv8

Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cε (PKCε), because Bv8 caused translocation of PKCε to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCε. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freunds adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.

Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1 In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O‐βglucosylated analogs ([Ser7‐O‐βGlc]dermorphin and [Ser7‐O‐βGlc(Ac)4]‐dermorphin) and C‐αgalactosylated analogs ([Ala7‐C‐αGal]dermorphin and [Ala7‐C‐αGal(Ac)4]‐dermorphin). 2 O‐ and C‐glycosylation of dermorphin halved the peptide affinity for brain μ‐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 μmol 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 μ‐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‐β‐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‐αgalactoside was about two times higher than that of the O‐βglucoside, excluding the implication of GLUT‐1 that is known to be selective for O‐β‐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.

Bv8, the amphibian homologue of the mammalian prokineticins, modulates ingestive behaviour in rats

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). 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. 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. 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. 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. 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.

Triazine compounds as antagonists at Bv8-prokineticin receptors.

On the basis of a Janssens patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential non peptidic prokineticin receptor antagonists, containing the following substitutions: (N(1) and N(5) link a 4-methoxybenzyl and a 4-ethylbenzyl, respectively; C(2) can link an amino-ethyl-guanidine (reference compound 1) or an ethylendiamine (2) or an amino-ethyl-amino-2-imidazoline (3). New compounds were assessed for PKR1 and PKR2 affinity. Antagonist properties were evaluated as inhibition of 1 nM Bv8-induced intracellular Ca2+ mobilization.

Biological activities of Bv8 analogues

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.

Pharmacology of Amphibian Opiate Peptides

In 1980 the skin of certain frogs belonging to the genus Phyllomedusinae was found to contain two new peptides that proved to be selective mu-opioid agonists, and named dermorphins. Since 1987 deltorphins, a family of highly selective delta-opioid peptides were identified either by cloning of the cDNA from frog skins or isolation of the peptides. The distinctive feature of opioid peptides is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe. The discovery of the amphibian opiate peptides, provided new insights into the functional role of the mu- and delta-opiate systems. It also provided models for novel analgesics with enhanced therapeutic benefits and reduced toxicity.

Chapter 11 Bv8/Prokineticins and their Receptors: A New Pronociceptive System

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) have been identified and linked to several biological effects as gut motility, neurogenesis, angiogenesis, circadian rhythms, hematopoiesis, and nociception. Emerging evidences indicated that prokineticins are also associated with pathologies of the reproductive and nervous system, myocardial infarction, and tumorigenesis. Bv8 elicits a dose-dependent reduction in nociceptive threshold to thermal, mechanical, and chemical stimuli. The prokineticin receptors are present in a fraction of C- and Adelta-fiber neurons also expressing the vanilloid receptors, TRPV1 and TRPA1. Mice lacking PKR genes exhibit impaired Bv8-induced hyperalgesia, develop deficient responses to noxious heat, capsaicin, and protons and show reduced thermal and mechanical hypersensitivity to paw inflammation, indicating a requirement for PKR signaling in activation and sensitization of primary afferent fibers. Bv8/PK2 is highly expressed by neutrophils and other inflammatory cells and must be considered as new pronociceptive mediators in inflamed tissues. Bv8-like hyperalgesic activity was demonstrated in extracts of rat inflammatory granulocytes. Bv8 stimulates macrophage and T lymphocyte to differentiate towards an inflammatory and Th1 profile indicating that Bv8/PK2 plays a role in immunoinflammatory responses. Blockade of PKRs may represent a novel therapeutic strategy in acute and inflammatory pain conditions.