Effect of the C-terminus of murine S100A9 protein on experimental nociception

Calcium-binding protein S100A9 induces antinociception in mice evaluated by the writhing test. Similarly, a peptide identical to the C-terminus of murine S100A9 (mS100A9p) inhibits the hyperalgesia induced by jararhagin, a metalloprotease. Thus, we investigated the effect of mS100A9p on different models used to evaluate nociception. mS100A9p induced a dose-dependent inhibitory effect on the writhing test, and on mechanical hyperalgesia induced by carrageenan. mS100A9p inhibited thermal hyperalgesia induced by carrageenan. mS100A9p did not modify the nociceptive response in hot plate or tail-flick tests. These data demonstrate that the C-terminus of S100A9 protein interferes with control mechanisms of inflammatory pain.     

Antinociceptive effect of the C-terminus of murine S100A9 protein on experimental neuropathic pain

The synthetic peptide identical to the C-terminus of murine S100A9 protein (mS100A9p) has antinociceptive effect on different acute inflammatory pain models. In this study, the effect of mS100A9p was investigated on neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. Hyperalgesia, allodynia, and spontaneous pain were assessed to evaluate nociception. These three signs were detected as early as 2 days after sciatic nerve constriction and lasted for over 14 days after CCI. Rats were treated with different doses of mS100A9p by intraplantar, oral, or intrathecal routes on day 14 after CCI, and nociception was evaluated 1h later. These three routes of administration blocked hyperalgesia, allodynia and spontaneous pain. The duration of the effect of mS100A9p depends on the route used and phenomenon analyzed. Moreover, intraplantar injection of mS100A9p in the contralateral paw inhibited the hyperalgesia on day 14 days after CCI. The results obtained herein demonstrate the antinociceptive effect of the C-terminus of murine S100A9 protein on experimental neuropathic pain, suggesting a potential therapeutic use for it in persistent pain syndromes, assuming that tolerance does not develop to mS100A9p.     

Antinociceptive Activity of the Ethanolic Extract,Fractions, and Aggregatin D Isolated from Sinningia aggregata Tubers

The present study investigated the effects of the ethanolic extract (ESa), fractions, and compounds isolated from Sinningia aggregata in male Swiss mice on carrageenan-induced paw edema, neutrophil migration, mechanical hyperalgesia, formalin-induced nociception, and lipopolysaccharide-induced fever. The ESa did not alter edema, neutrophil migration, or fever at any of the doses tested. However, the ESa reduced phase II of formalin-induced nociception and carrageenan-induced mechanical hyperalgesia. The petroleum ether (PE) and ethyl acetate (EA) fractions and aggregatin D (AgD; isolated from the EA fraction) reduced formalin-induced nociception. Anthraquinones from the PE fraction were ineffective. AgD also inhibited carrageenan-induced mechanical hyperalgesia. Neither the ESa nor AgD altered thermal nociception or motor performance. Local administration of AgD also reduced hyperalgesia induced by carrageenan, bradykinin, tumor necrosis factor-α, interleukin-1β, cytokine-induced neutrophil chemoattractant, prostaglandin E₂, and dopamine but not hyperalgesia induced by forskolin or dibutyryl cyclic adenosine monophosphate. The positive control dipyrone reduced the response induced by all of the stimuli. Additionally, glibenclamide abolished the analgesic effect of dipyrone but not the one induced by AgD. AgD did not change lipopolysaccharide-induced nitric oxide production by macrophages or the nociception induced by capsaicin, cinnamaldehyde, acidified saline, or menthol. These results suggest that the ESa has important antinociceptive activity, and this activity results at least partially from the presence of AgD. AgD reduced mechanical hyperalgesia induced by several inflammatory mediators through mechanisms that are different from classic analgesic drugs.     

Antinociceptive, antiedematogenic and antiangiogenic effects of benzaldehyde semicarbazone

Semicarbazones induce an anticonvulsant effect in different experimental models. As some anticonvulsant drugs also have anti-inflammatory activity, the effects of benzaldehyde semicarbazone (BS) on models of nociception, edema and angiogenesis were investigated. BS (10, 25 or 50 mg/kg, i.p.) markedly inhibited the second phase of nociceptive response induced by formaldehyde (0.34%, 20 microl) in mice, but only the highest dose inhibited the first phase of this response. The thermal hyperalgesia and mechanical allodynia induced by carrageenan (1%, 50 microl, i.pl.) in rats were also inhibited by BS (50 mg/kg, i.p.). However, treatment of mice with BS did not induce an antinociceptive effect in the hot-plate model. The paw edema induced by carrageenan (1%, 50 microl, i.pl.) in rats was inhibited by BS (25 or 50 mg/kg, i.p.). Treatment of mice with BS (0.25, 0.5 or 2.5 mg/kg/day, i.p., 7 days) also inhibited angiogenesis induced by subcutaneous implantation of a sponge disc. It is unlikely that the antinociceptive effect induced by BS results from motor incoordination or a muscle relaxing effect, as the mice treated with this drug displayed no behavioral impairment in the rotarod apparatus. In conclusion, we demonstrated that BS presents antinociceptive, antiedematogenic and antiangiogenic activities. An extensive investigation of the pharmacological actions of BS and its derivatives is justified and may lead to the development of new clinically useful drugs.     

Activation of transient receptor potential ankyrin 1 evokes nociception through substance P release from primary sensory neurons

To examine mechanisms underlying substance P (SP) release from primary sensory neurons in response to activation of the non-selective cation channel transient receptor potential ankyrin 1 (TRPA1), SP release from cultured rat dorsal root ganglion neurons was measured, using radioimmunoassay, by stimulating TRPA1 with allyl isothiocyanate (AITC), a TRPA1 agonist. AITC-evoked SP release occurred in a concentration- and time-dependent manner. Interestingly, p38 mitogen-activated protein kinase (p38) inhibitor SB203580 significantly attenuated AITC-evoked SP release. The in vivo effect of AITC-evoked SP release from primary sensory neurons in mice was evaluated. Hind paw intraplantar injection of AITC induced nociceptive behaviors and inflammation (edema, thermal hyperalgesia). AITC-induced thermal hyperalgesia and edema were inhibited by intraplantar pre-treatment with either SB203580 or neurokinin-1 receptor antagonist CP96345. Moreover, intrathecal pre-treatment with either CP96345 or SB203580 inhibited AITC-induced nociceptive behaviors and thermal hyperalgesia. Immunohistochemical studies demonstrated that intraplantar AITC injection induced the phosphorylation of p38 in mouse dorsal root ganglion neurons containing SP. These findings suggest that activation of TRPA1 evokes SP release from the primary sensory neurons through phosphorylation of p38, subsequent nociceptive behaviors and inflammatory responses. Furthermore, the data also indicate that blocking the effects of TRPA1 activation at the periphery leads to significant antinociception.     

Anti-edematogenic effects of velutinol A isolated from Mandevilla velutina: evidence for a selective inhibition of kinin B1 receptor-mediated responses

This study assesses the effects of compound velutinol A obtained from M. velutina in the rat paw edema induced by several phlogistic agents. Attempts were made to analyze how velutinol A is able to inhibit kinin B(1) receptor-mediated inflammatory responses. Velutinol A (100 nmol/paw) partially reduced (about 30%) the edema evoked by carrageenan (300 microg/paw). However, velutinol A (100 nmol/paw) failed to affect the edema induced by histamine (200 nmol/paw), substance P (30 nmol/paw), PAF (10 nmol/paw) or BK (3 nmol/paw). Interestingly, the edema caused by the selective kinin B(1) receptor agonist des-Arg(9)-BK (100 nmol/paw) in animals pre-treated with PAF or LPS was significantly inhibited by velutinol A (100 nmol/paw) (48 and 46%, respectively). A similar inhibition of des-Arg(9)-BK-induced edema after pre-treatment with PAF was obtained with the non-peptidic and selective B(1) receptor antagonist SSR 240612 (60 nmol/paw) (46%). In addition, the systemic administration of velutinol A (10 mg/kg, i.p.) or SSR 240612 (1 mg/kg, i.p.) also caused a significant reduction of des-Arg(9)-BK (100 nmol/paw)-induced edema in PAF-treated rats (51 and 43%, respectively). The results provide convincing evidence that velutinol A selectively blocks the edema responses mediated by B(1) receptor activation in vivo. This compound might represent a new non-peptidic and selective antagonist for kinin B(1) receptors.     

Biochemical characterization of the murine S100A9 (MRP14) protein suggests that it is functionally equivalent to its human counterpart despite its low degree of sequence homology.

Due to the low degree of sequence similarity it has been speculated that murine and human S100A9 (MRP14), an inflammatory marker protein belonging to the S100 protein family, may have different cellular functions in mouse and man. The present study was undertaken to investigate the murine S100A9 protein (mS100A9) biochemically. We demonstrate that in murine peripheral CD11b+ cells up to 20% of the protein of the cytosolic fraction consists of mS100A9 and that several minor mS100A9 isoforms are present. Cell fractionation experiments with CD11b+ murine leukocytes showed that mS100A9 is found in the cytosol as well as in the insoluble fraction. Transient expression of a green fluorescence protein-mS100A9 fusion in mammalian cells revealed that mS100A9 is localized in neither the nucleus nor the vesicles. Recombinantly expressed murine S100A9 interacts in vitro with murine and human S100A8 in an in vitro glutathione S-transferase pull-down assay. Homodimerization was not observed. For further biochemical analysis the myeloid 32D cell line is presented as a suitable model, to study murine myeloid expressed S100 proteins. Both murine S100A9 and its dimerization partner mS100A8 are expressed at the onset of granulocyte-colony stimulating factor induced myeloid differentiation. Substantial amounts of this complex are constitutively secreted by granulocytic 32D cells into the medium. In summary, these data suggest, that the human and murine S100A9 may share a higher degree of functional homology than of sequence similarity.     

Pathway to carrageenan-induced inflammation in the hind limb of the rat

A sequential 43-step pathway scheme for the inflammatory response of the rat to interdermal injection of carrageenan (C) was devised. It consisted of a nonphagocytic inflammatory response (NPIR) followed by a phagocytic inflammatory response (PIR) in the dermis and an epidermal NPIR. The dermal NPIR comprised edema, hyperemia, and hyperalgesia followed by hypoalgesia. Antiserotonin agents inhibited the hypoalgesia and part of the edema. These findings and histological observations suggested that dermal mast cells were injured by C. The hyperalgesia and part of the edema were sensitive to arachidonate cyclooxygenase inhibitors (AACOIs). It is speculated that injured mast cells metabolize arachidonic acid and reactive intermediates, not prostaglandins, mediate the NPIR hyperalgesia and part of the edema. The dermal PIR consisted of mobilization of neutrophils, edema, hyperalgesia, mobilization of monocytes, and proliferation of fibroblasts and vascular tissue. Selective drug actions revealed that the edema, hyperalgesia, and monocyte mobilization of the PIR depended on the mobilization of neutrophils. After the mobilization of neutrophils, AACOIs reduced edema formation and hyperalgesia. Arachidonic acid metabolism by neutrophils is speculated to produce the mediators of phagocytic inflammatory (PI) edema and hyperalgesia. Monocyte function was associated with cessation of PI edema formation and phagocytosis of neutrophils and cellular debris. Interleukin 1 is speculated to mediate the adherence of neutrophils to injured dermal endothelium. The epidermal NPIR consisted of edema, hyperplasia, and hyperkeratosis. These parameters were not studied mechanistically. There was no evidence for histamine, bradykinin, platelets, clotting factors, or complement mediating any events in the pathway.     

Jararhagin,a snake venom metalloproteinase,induces a specialized form of apoptosis (anoikis) selective to endothelial cells

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40μg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-x_L. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.     

Structure/function studies of S100A8/A9

Summary The functional importance of members of the S100 Ca²⁺-binding protein family is recently emerging. A variety of activities, several of whcih are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10⁻¹³-10⁻¹¹ M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.     

Structure/function studies of S100A8/A9

The functional importance of members of the S100 Ca²⁺-binding protein family is recently emerging. A variety of activities, several of which are apparently opposing, are attributed to S100A8, a protein implicated in embryogenesis, growth, differentiation, and immune and inflammatory processes. Murine (m) S100A8 was initially described as a chemoattractant (CP-10) for myeloid cells. It is coordinately expressed with mS100A9 (MRP14) in neutrophils and the non-covalent heterodimer is presumed to be the functional intracellular species. The extracellular chemotactic activity of mS100A8, however, is not dependent on mS100A9 and occurs at concentrations (10^-13–10^-11 M) at which the non-covalent heterodimer would probably dissociate. This review focuses on the structure and post-translational modifications of mS100A8/A9 and their effects on function, particularly chemotaxis.     

S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/ A9 treatment increased migration and invasionat lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogenactivated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPKdependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.     

Antinociceptive effect of Nidularium procerum: a Bromeliaceae from the Brazilian coastal rain forest

Nidularium procerum, a common plant of the Brazilian flora, has not yet been studied for its pharmacological properties. We report here that extracts of N. procerum show both analgesic and anti-inflammatory properties. Oral (p.o.) or intraperitoneal (i.p.) administration of an aqueous crude extract from leaves of N. procerum (LAE) inhibited the writhing reaction induced by acetic acid (ED50 value = 0.2 mg/kg body weight, i.p.) in a dose-dependent manner. This analgesic property was confirmed in rats using two different models of bradykinin-induced hyperalgesia; there was 75% inhibition of pain in the modified Hargreaves assay, and 100% inhibition in the classical Hargreaves assay. This potent analgesic effect was not blocked by naloxone, nor was it observed in the hot plate model, indicating that the analgesic effect is not associated with the activation of opioid receptors in the central nervous system. By contrast, we found that LAE (0.02 microg/ml) selectively inhibited prostaglandin E2 production by cyclooxygenase (COX)-2, but not COX-1, which is a plausible mechanism for the analgesic effect. A crude methanol extract from the leaves also showed similar analgesic activity. An identical extract from the roots of N. procerum did not, however, block acetic acid-induced writhes, indicating that the analgesic compounds are concentrated in the leaves. Finally, we found that LAE inhibited an inflammatory reaction induced by lipopolysaccharide in the pleural cavity of mice.     

Anti-inflammatory activity of licochalcone A isolated from Glycyrrhiza inflata

Licochalcone A was isolated from the roots of Glycyrrhiza inflata and evaluated for its anti-inflammatory activity in xylene-induced mice ear edema and carrageenan-induced paw edema tests. At the same time, the inhibition of prostaglandin biosynthesis by licochalcone A was also studied in lipopolysaccharide (LPS)-induced mouse macrophage cells. At 5 mg/ ear, licochalcone A showed remarkable effects against acute inflammation induced by xylene, and at the doses of 2.5, 5, 10 mg/kg (p.o.), licochalcone A reduced significantly paw edema induced by carrageenan compared to the control at the fourth hour. Both COX-2 activity and expression were significantly inhibited by licochalcone A at all the test doses. Therefore, licochalcone A could be a useful compound for the development of new anti-inflammatory agents.     

The S100A8/A9 heterodimer amplifies proinflammatory cytokine production by macrophages via activation of nuclear factor kappa B and p38 mitogen-activated protein kinase in rheumatoid arthritis

S100A8 and S100A9, two Ca²⁺-binding proteins of the S100 family, are secreted as a heterodimeric complex (S100A8/A9) from neutrophils and monocytes/macrophages. Serum and synovial fluid levels of S100A8, S100A9, and S100A8/A9 were all higher in patients with rheumatoid arthritis (RA) than in patients with osteoarthritis (OA), with the S100A8/A9 heterodimer being prevalent. By two-color immunofluorescence labeling, S100A8/A9 antigens were found to be expressed mainly by infiltrating CD68⁺ macrophages in RA synovial tissue (ST). Isolated ST cells from patients with RA spontaneously released larger amounts of S100A8/A9 protein than did the cells from patients with OA. S100A8/A9 complexes, as well as S100A9 homodimers, stimulated the production of proinflammatory cytokines, such as tumor necrosis factor alpha, by purified monocytes and in vitro-differentiated macrophages. S100A8/A9-mediated cytokine production was suppressed significantly by p38 mitogen-activated protein kinase (MAPK) inhibitors and almost completely by nuclear factor kappa B (NF-κB) inhibitors. NF-κB activation was induced in S100A8/A9-stimulated monocytes, but this activity was not inhibited by p38 MAPK inhibitors. These results indicate that the S100A8/A9 heterodimer, secreted extracellularly from activated tissue macrophages, may amplify proinflammatory cytokine responses through activation of NF-κB and p38 MAPK pathways in RA.     

Calotropis procera latex-induced inflammatory hyperalgesia--effect of antiinflammatory drugs

The milky white latex of plant Calotropis procera produces inflammation of the skin and mucous membranes on accidental exposure. It produces edema on local administration due to the release of histamine and prostaglandins and is associated with hyperalgesia. In the present study we have evaluated the antiedematous and analgesic activity of antiinflammatory drugs against inflammatory response induced by dried latex (DL) of C procera in rat paw edema model. An aqueous extract of DL of C procera was injected into the subplantar surface of the rat paw and the paw volume was measured by a plethysmometer at 0, 1, 2, 6, 12, and 24 hours. Concomitantly the hyperalgesic response was also evaluated by motility test, stair climbing ability test, dorsal flexion pain test, compression test, and observing the grooming behavior. The inhibitory effect of diclofenac and rofecoxib on edema formation and hyperalgesic response was compared with cyproheptadine (CPH). DL-induced edema formation was maximum at 2 hours that was associated with decreased pain threshold, functional impairment, and grooming. Treatment with antiinflammatory drugs and CPH significantly attenuated the edematous response and grooming, increased the pain threshold, and improved functional parameters. Both antiinflammatory and antiserotonergic drugs significantly inhibited the hyperalgesia associated with DL-induced paw edema. Rofecoxib was found to be superior than diclofenac and was as effective as CPH in ameliorating the hyperalgesia. However, it was found to be less effective than CPH in attenuating edema formation.     

The role of sensorial neuropeptides in the edematogenic responses mediated by B(1) agonist des-Arg(9)-BK in rats pre-treated with LPS

In the present study we have investigated some of the mechanisms underlying B(1) kinin receptor-induced paw edema formation in rats that had been treated with LPS, paying special attention to the involvement of neurogenic inflammation. Intradermal (i.d.) injection of the B(1) receptor agonist des-Arg(9)-BK (100 nmol/paw) resulted in a marked increase in paw volume in animals pre-treated with LPS (0.40+/-0.06 ml). The co-injection of the selective NK(1) FK888 (1 nmol/paw) or NK(2) SR 48968 (3 nmol/paw) receptor antagonists resulted in a significant inhibition of the edema induced by des-Arg(9)-BK (30+/-4 and 25+/-7%, respectively). The NK(3) SR 142801 (3 nmol/paw) antagonist did not demonstrate any significant effect on B(1) receptor-mediated paw edema. The edema induced by des-Arg(9)-BK was also significantly inhibited (33+/-5%) by the co-injection of the CGRP-receptor antagonist CGRP 8-37 (1 nmol/paw) or by treatment of animals with capsaicin (50 mgkg(-1), s.c., 48 h, prior) (45+/-4%). The pre-treatment of animals with methysergide or with mianserin, 5-HT(1) and 5HT(2) antagonists, respectively (both 10 mgkg(-1), i.p. 30 min), resulted in a significant reduction of the edema mediated by B(1) receptors (23+/-5 and 20+/-3%, respectively). In addition, compound 48/80 (12 microg/paw, 24 h) significantly reduced des-Arg(9)-induced paw edema in rats pre-treated with LPS (23+/-3%), while the treatment of animals with the H(1) receptor antagonist pyrilamine (10 mgkg(-1), i.p., 30 min) failed to affect the edematogenic responses involving B(1) receptors. Finally, the co-injection of NOS inhibitors L-NAME (100 nmol/paw) or 7-NINA (10 nmol/paw) did not affect the rat paw edema caused by des-Arg(9)-BK, whereas they significantly inhibited BK-induced paw edema. Jointly, the results of the present study show that the edematogenic response mediated by the activation of B(1) receptors, in animals pre-treated with LPS, involves the release of tachykinins and CGRP, as well as serotonin, while NO and histamine seem not to be involved. Therefore, these data further support the notion that B(1) receptors have an important role in modulating the inflammatory processes.     

Evidence for a spinal serotonergic control of the peripheral inflammation in the rat

We investigated the effect of serotonergic agonists and antagonists injected intrathecally by direct punction of the spinal cord at the lumbar level (between L5-L6) on peripheral inflammatory edema. Edema was induced by carrageenan injected subcutaneously in one hindpaw 30 min after spinal treatments. Serotonin (0.1, 1, 10 pmol) caused a graded-inhibition of the inflammatory paw edema. The corticosteroid inhibitor aminoglutethimide (100 mg/kg, p.o. 1.5 h before spinal treatment) did not modify this effect. The 5-HT1A agonist buspirone and the 5-HT1B/1D agonist sumatriptan (0.1, 1.0 and 10 nmol) also inhibited paw edema. The 5-HT1,2 antagonist methysergide (10 and 100 pmol) enhanced edema, but higher doses ( 4 and 8 nmol) diminished edema. NAN-190 (5-HT1 antagonist; 1 and 10 nmol) increased paw edema, while ritanserin (5-HT2 antagonist; 1 nmol) inhibited paw edema. Ondansetron (5-HT3 antagonist; up to 10 nmol) did not affect edema, but metoclopramide (5-HT3 antagonist / 5-HT4 agonist; 5, 10 and 30 pmol) inhibited edema. These data suggest that a tonic release of serotonin in the spinal cord may occurs during ongoing peripheral inflammation, modulating the neurogenic component of edema either by an inhibitory action on 5-HT1 receptors or by a stimulatory action on 5-HT2 receptors. A disfunction in such mechanism may be involved in the pathophysiology of certain types of headaches or migraine, which seem to depend on neurogenic vasodilation, and may also help to explain the therapeuthic effectiveness of some serotonergic agents in these conditions.     

Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.