Lucia Negri

Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for δ opioid receptors

With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for delta opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2.

Bv8, a small protein from frog skin and its homologue from snake venom induce hyperalgesia in rats

From skin secretions of Bombina variegata and Bombina bombina, we isolated a small protein termed Bv8. The sequence of its 77 amino acids was established by peptide analysis and by cDNA cloning of the Bv8 precursor. Bv8 stimulates the contraction of the guinea-pig ileum at nanomolar concentrations. The contraction is not inhibited by a variety of antagonists. Injection of a few micrograms of Bv8 into the brain of rats elicits, as assessed by the tail-flick test and paw pressure threshold, a marked hyperalgesia which lasts for about 1 h. Bv8 is related to protein A, a component of the venom of the black mamba. After i.c.v. injection, protein A is even more active than Bv8 in inducing hyperalgesia.

RELATIVE POTENCY OF BOMBESIN‐LIKE PEPTIDES

1 The pharmacological activity of two natural bombesin‐like peptides, alytesin and litorin, and 25 related synthetic peptides has been compared to that of bombesin. 2 The minimum length of the amino acid chain required for the first appearance of bombesin‐like effects was represented by the C‐terminal heptapeptide, and the minimum length for maximal effects by the C‐terminal nonapeptide. The latter possessed approximately the same activity as bombesin and may be considered a good substitute. 3 Both the tryptophan and histidine residues seemed to be essential for bombesin‐like activity. 4 The C‐terminal octapeptide was less active than either bombesin or the C‐terminal nonapeptide and its action was more rapid in onset and less sustained. 5 Litorin apparently has an intermediate position between bombesin octapeptide and bombesin nonapeptide in the speed and duration of its effects. The relationship between structure and activity is discussed.

Occurrence of bombesin and alytesin in extracts of the skin of three European discoglossid frogs and pharmacological actions of bombesin on extravascular smooth muscle

1 Methanol extracts of the skin of Bombina bombina and Bombina variegata variegaia, two European discoglossid frogs, contain an active tetradecapeptide, bombesin. Alytesin, a tetradecapeptide strictly related to bombesin is present in extracts of the skin of Alytes obstetricans, another European discoglossid frog. The American frog Rana pipiens, contains in its skin ranatensin, an endecapeptide related to bombesin and alytesin. 2 Passage of crude skin extracts of Bombina through a column of alumina yields eluates which may be considered free of other peptide contaminants and are suitable for the isolation of bombesin in a pure form. 3 Bombesin has a stimulant action on several preparations of intestinal, uterine and urinary tract smooth muscle. Sometimes the effect is easily repeatable and shows a fair proportionality to the dose, but at other times a prompt and intense tachyphylaxis is observed. Other smooth muscle preparations are poorly sensitive or insensitive to bombesin. The rat uterus, the kitten small intestine, the guinea‐pig colon and the rat urinary bladder may be used for the quantitative bioassay of bombesin. 4 Bombesin‐like peptides may easily be distinguished from all other naturally occurring peptides by parallel assay. They constitute a new group of active peptides possessing a peculiar spectrum of activity.

The brain-gut-skin triangle: New peptides

New data on tachykinins and bombesins are displayed and the present situation of research on the novel amphibian skin peptides sauvagine and dermorphin is illustrated. The potent stimulant effect of sauvagine on ACTH and beta-endorphin release has been confirmed both in vivo and on columns of isolated and dispersed rat pituitary cells, and similarly the potent inhibitory effect on PRL and GH release, both in the rat and man. Particular emphasis is laid on the occurrence of sauvagine-like immunoreactivity in fish urophysis and in amphibian nervous structures, including the retina. It is suggested that the long-searched corticotropin releasing factor and PRL release-inhibiting factor may be a sauvagine-like peptide. Dermorphin, in its turn, has been found to cause, by intracerebroventricular injection, not only analgesia and catalepsy, but also conspicuous EEG and behavioral changes in the rabbit and chick, as well as a sharp reduction in gastric emptying time and gastric acid output in the rat, together with marked stimulation of PRL release.

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.

Bv8, the amphibian homologue of the mammalian prokineticins, induces a proinflammatory phenotype of mouse macrophages

The small protein Bv8, isolated from the amphibian skin, belongs to a novel family of secreted proteins linked to several biological effects. We describe the expression of Bv8/prokineticins and their receptors in mouse macrophages, and characterize their proinflammatory activities. The rodent analogue of Bv8, prokineticin‐2, is expressed by macrophages, as well as its G‐protein‐coupled receptor prokineticin receptor (PKR‐1 and PKR‐2). PKR‐1 is expressed more abundantly. Bv8 induces potent chemotaxis of macrophages at concentrations as low as 10−12 M. It stimulates lipopolysaccharide‐induced production of the proinflammatory cytokines IL‐1 and IL‐12, reducing that of the anti‐inflammatory cytokine IL‐10. The effects are observed starting at the very low concentration of 10−11 M. Effects on chemotaxis and cytokine are not pertussis‐toxin sensitive, but are completely prevented by addition of the phospholipase inhibitor U73122, suggesting a Gq protein is involved in the Bv8‐induced effects. Studies in PKR‐1 knockout mice indicate that all the activities exerted by Bv8 on macrophages are mediated by the PKR‐1 receptor. In conclusion, Bv8 appears to be able to induce the macrophage to migrate and to acquire a proinflammatory phenotype.

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.