Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells.

BACKGROUND Avicularin is a plant-derived flavonoid used in traditional Chinese medicine to treat conditions that include ankle fracture. Bradykinin stimulated MG-63 human osteoblastic osteosarcoma cells has previously been studied in an in vitro model. This study aimed to investigate the effects of avicularin on bradykinin-treated MG-63 human osteoblastic osteosarcoma cells in vitro. MATERIAL AND METHODS MG-63 cells were treated with increasing concentrations of avicularin for 48 hours, followed by 1 µM of bradykinin for 24 h. The MTT assay was used to measure cell viability. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the expression of inflammatory mediators, interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-alpha) mRNA and protein, respectively. The expression of oxidative stress factors, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase were measured. Western blot and qRT-PCR were performed to determine p38, p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein levels and mRNA expression, respectively. RESULTS Avicularin had no cytotoxic effect on MG-63 cells. Avicularin significantly upregulated the expression levels IL-1ß, IL-6, and TNF-alpha in the bradykinin treated group in a dose-dependent manner. Avicularin reduced the level of MDA and the activity of SOD and catalase in the bradykinin treated MG-63 cells, reduced p-p38, p-p65, iNOS, and COX-2 expression, and decreased the p-p38/p38 ratio and the p-p65/p65 ratio in bradykinin treated MG-63 cells in a dose-dependent manner. CONCLUSIONS Avicularin reduced the expression of inflammatory cytokines and the levels of markers of oxidative stress in MG-63 human osteoblastic osteosarcoma cells in vitro.

Effects of bradykinin on the survival of multiterritory perforator flaps in rats.

BACKGROUND: Bradykinin, a vasoactive peptide, has many biological functions. For example, it accelerates angiogenesis. Thus, we studied the effects of bradykinin on the survival of perforator flaps. METHODS: Averagely, 50 male Sprague-Dawley rats were divided into control and bradykinin groups and underwent procedures to the multiterritory perforator flap. Areas of flap survival were tested 7 days later. Flap perfusion was evaluated by laser Doppler imaging. We assessed the extent of autophagy by determining LC3-II/I, Beclin 1, and p62. Flap angiogenesis was assessed by immunohistochemistry and H&E staining. We measured the level of vascular endothelial growth factor (VEGF) protein using western blot. We assessed oxidative stress by measuring the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) levels. The apoptotic index was also evaluated by western blot, and we determined nitric oxide (NO) production using an NO assay kit. RESULTS: The bradykinin group exhibited significantly larger areas of flap survival, higher blood supply, and more neovascularization. The bradykinin group also had higher SOD activity, higher VEGF expression and NO content, and reduced MDA compared to the control group. Rats treated with bradykinin also had lower levels of apoptosis and autophagy relative to the control group. CONCLUSION: Our results suggest that bradykinin promotes the survival of multiterritory perforator flaps by increasing angiogenesis, promoting the release of NO, suppressing apoptosis, reducing oxidative stress, and inhibiting autophagy.

Tambulin is a major active compound of a methanolic extract of fruits of Zanthoxylum armatum DC causing endothelium-independent relaxations in porcine coronary artery rings via the cyclic AMP and cyclic GMP relaxing pathways.

BACKGROUND: Zanthoxylum armatum DC (Z. armatum), belonging to Rutaceae family, has been traditionally used for the treatment of various diseases such as hypertension, abdominal pain, headache, fever, high altitude sickness, diarrhea, dysentery, and as a tonic, condiment, and an anthelmintic treatment. HYPOTHESIS: The present study aims to evaluate the vasorelaxant effect of a methanolic extract of the fruits of Z. armatum, isolate the active components and characterize the underlying mechanism. STUDY DESIGN: A methanolic extract of fruits of Z. armatum was prepared and its vasorelaxant effect was studied using porcine coronary artery rings. Thereafter, the methanolic extract was analyzed, and a major compound was isolated and its structure elucidated (tambulin). Different pharmacological tools were used to characterize the vasorelaxant effect of tambulin. RESULTS: The methanolic extract and the isolated tambulin caused similar endothelium-independent relaxations of porcine coronary artery rings with and without endothelium indicating a direct relaxing effect at the vascular smooth muscle. Tambulin did not affect the relaxation curves to the endothelium-dependent vasodilators, bradykinin and the calcium ionophore A23187 in rings with endothelium. Tambulin (1 µM) slightly but significantly shifted leftwards the concentration-relaxation curve to the endothelium-independent vasodilators, sodium nitroprusside (SNP), forskolin (FC) and isoproterenol but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41-2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with tambulin inhibited, in a concentration-dependent manner, contractions to KCl, serotonin (5-HT), CaCl2 and U46619 in coronary artery rings without endothelium. Both the protein kinase A (H-89, 10 µM) and the protein kinase G (Rp-8-br-cyclic GMPS, 30 µM) inhibitors significantly reduced relaxations to tambulin in coronary artery rings without endothelium. CONCLUSION: The present findings indicate that tambulin isolated from Z. armatum (fruits) is a major active principle inducing vasorelaxation through a direct effect at the vascular smooth muscle and involving both the cyclic AMP and/or cyclic GMP relaxing pathways.

Effect of rosmarinic acid on the arterial blood pressure in normotensive and hypertensive rats: Role of ACE.

BACKGROUND: In recent years, it has been demonstrated the inhibitory effect of some plant species on the angiotensin-converting enzyme and rosmarinic acid is a prominent constituent of these species. HYPOTHESIS/PURPOSE: This study was carried out to verify the effect of rosmarinic acid on blood pressure through inhibitory activity on angiotensin-converting enzyme in rats. STUDY DESIGN: The arterial hypertension was promoted using 2-kidneys 1-clip model in rats. The potential inhibitory rosmarinic acid effect on angiotensin-converting enzyme activity was compared with captopril actions by analyzing in vivo blood pressure dose-response curves to angiotensin I and bradykinin. The in vitro plasma angiotensin-converting enzyme activity was measured by fluorimetry using the substrate Abz-FRK(Dnp)P-OH substrate. In addition, dosages of nitrite/nítrate analysis were carried out. RESULTS: (1) rosmarinic acid caused systolic blood pressure dose-dependent decrease in hypertensive rats; (2) The angiotensin I dose-response curves demonstrated that rosmarinic acid promotes minor changes in systolic blood pressure only in the hypertensive group; (3) The bradykinin dose-response curves showed that both rosmarinic acid and captopril promoted a systolic blood pressure reduction, but only the captopril effect was significant; (4) The angiotensin-converting enzyme activity in rat lung tissue was inhibited by the rosmarinic acid in a dose dependent manner; (5) The analysis of nitrite/nítrate plasma concentrations showed no significant difference among the experimental groups. CONCLUSION: The rosmarinic acid is effective in reducing blood pressure, selectively, only in hypertensive animals. The rosmarinic acid (173µM) promoted almost a 98.96% reduction on angiotensin-converting enzyme activity.

NG25, an inhibitor of transforming growth factor­ß­activated kinase 1, ameliorates neuronal apoptosis in neonatal hypoxic­ischemic rats.

Transforming growth factor (TGF)­ß­activated kinase 1 (TAK1) was found to be activated by TGF­ß and acts as a central regulator of cell death in various types of disease. However, the expression and function of TAK1 in the neonatal brain following hypoxia­ischemia (HI) remains unclear. In the present study, western blotting and immunofluorescence were employed to determine the expression and distribution of TAK1 in the brain cortex of a perinatal HI rat model. In addition, the specific inhibitor of TAK1, NG25 was administered via intracerebroventricular injection, prior to insult of the neonatal rat brains, for neuroprotection. Western blotting and double immunofluorescence indicated that an increased expression level of phosphorylated­TAK1 was observed, and was localized with neurons and astrocytes, compared with the sham group. Further study demonstrated that injection of NG25 prior to insult significantly inhibited TAK1/c­Jun N­terminal kinases activity and dramatically ameliorated acute hypoxic­ischemic cerebral injury by inhibiting cell apoptosis in perinatal rats. Thus, NG25 ameliorates neuronal apoptosis in neonatal HI rats by inhibiting TAK1 expression and cell apoptosis. In addition, NG25 may serve as a promising novel neuroprotective inhibitor for perinatal cerebral injury.

Ex vivo study of human visceral nociceptors.

OBJECTIVE: The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation. DESIGN: Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses. RESULTS: Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (-20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (-34.9±10.0%, n=7, p<0.05), a TRPV4 antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity. CONCLUSIONS: Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Extracts of Crataegus oxyacantha and Rosmarinus officinalis Attenuate Ischemic Myocardial Damage by Decreasing Oxidative Stress and Regulating the Production of Cardiac Vasoactive Agents.

Numerous studies have supported a role for oxidative stress in the development of ischemic damage and endothelial dysfunction. Crataegus oxyacantha (Co) and Rosmarinus officinalis (Ro) extracts are polyphenolic-rich compounds that have proven to be efficient in the treatment of cardiovascular diseases. We studied the effect of extracts from Co and Ro on the myocardial damage associated with the oxidative status and to the production of different vasoactive agents. Rats were assigned to the following groups: (a) sham; (b) vehicle-treated myocardial infarction (MI) (MI-V); (c) Ro extract-treated myocardial infarction (MI-Ro); (d) Co extract-treated myocardial infarction (MI-Co); or (e) Ro+Co-treated myocardial infarction (MI-Ro+Co). Ro and Co treatments increased total antioxidant capacity, the expression of superoxide dismutase (SOD)-Cu2+/Zn2+, SOD-Mn2+, and catalase, with the subsequent decline of malondialdehyde and 8-hydroxy-2'-deoxyguanosine levels. The extracts diminished vasoconstrictor peptide levels (angiotensin II and endothelin-1), increased vasodilators agents (angiotensin 1-7 and bradikinin) and improved nitric oxide metabolism. Polyphenol treatment restored the left intraventricular pressure and cardiac mechanical work. We conclude that Ro and Co treatment attenuate morphological and functional ischemic-related changes by both an oxidant load reduction and improvement of the balance between vasoconstrictors and vasodilators.

Bradykinin/B2 receptor activation regulates renin in M-1 cells via protein kinase C and nitric oxide.

In the collecting duct (CD), the interactions of renin angiotensin system (RAS) and kallikrein-kinin system (KKS) modulate Na+ reabsorption, volume homeostasis, and blood pressure. In this study, we used a mouse kidney cortical CD cell line (M-1 cells) to test the hypothesis that in the CD, the activation of bradykinin B2 receptor (B2R) increases renin synthesis and release. Physiological concentrations of bradykinin (BK) treatment of M-1 cells increased renin mRNA and prorenin and renin protein contents in a dose-dependent manner and increased threefold renin content in the cell culture media. These effects were mediated by protein kinase C (PKC) independently of protein kinase A (PKA) because B2R antagonism with Icatibant and PKC inhibition with calphostin C, prevented these responses, but PKA inhibition with H89 did not modify the effects elicited by the B2R activation. BK-dependent stimulation of renin gene expression in CD cells also involved nitric oxide (NO) pathway because increased cGMP levels and inhibition of NO synthase with L-NAME prevented it. Complementary renin immunohistochemical studies performed in kidneys from mice with conventional B2R knockout and conditional B2R knockout in the CD, showed marked decreased renin immunoreactivity in CD, regardless of the renin presence in juxtaglomerular cells in the knockout mice. These results indicate that the activation of B2R increases renin synthesis and release by the CD cells through PKC stimulation and NO release, which support further the interactions between the RAS and KKS.

Identification of multi-targeted anti-migraine potential of nystatin and development of its brain targeted chitosan nanoformulation.

The complex pathophysiology involved in migraine necessitates the drug treatment to act on several receptors simultaneously. The present investigation was an attempt to discover the unidentified anti-migraine activity of the already marketed drugs. Shared featured pharmacophore modeling was employed for this purpose on six target receptors (ß2 adrenoceptor, Dopamine D3, 5HT1B, TRPV1, iGluR5 kainate and CGRP), resulting in the generation of five shared featured pharmacophores, which were further subjected to virtual screening of the ligands obtained from Drugbank database. Molecular docking, performed on the obtained hit compounds from virtual screening, indicated nystatin to be the only active lead against the receptors iGluR5 kainate receptor (1VSO), CGRP (3N7R), ß2 adrenoceptor (3NYA) and Dopamine D3 (3PBL) with a high binding energy of -11.1, -10.9, -10.2 and -12kcal/mole respectively. The anti-migraine activity of nystatin was then adjudged by fabricating its brain targeted chitosan nanoparticles. Its brain targeting efficacy, analyzed qualitatively by confocal laser scanning microscopy, demonstrated a significant amount of drug reaching the brain. The pharmacodynamic models on Swiss male albino mice revealed significant anti-migraine activity of the nanoformulation. The present study reports for the first time the therapeutic potential of nystatin in migraine management, hence opening avenues for its future exploration.

Evaluation of Thr6-bradykinin purified from Polybia occidentalis wasp venom in the choline uptake of mammal cortices.

CONTEXT: Thr6-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals. OBJECTIVE: As such, the objective of this study was to evaluate the effect of Thr6-bradykinin on the cholinergic system of rats. MATERIALS AND METHODS: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [14C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr6-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000 µM). RESULTS: Data revealed that neither BK nor Thr6-BK at any of the six concentrations tested (from 0.039 to 40.000 µM) affected [14C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr6-BK did not disrupt the synaptosomes. DISCUSSION AND CONCLUSION: In contrast to previous reports for the insect central nervous system (CNS), Thr6-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.

Paraventricular Nucleus Modulates Excitatory Cardiovascular Reflexes during Electroacupuncture.

The paraventricular nucleus (PVN) regulates sympathetic outflow and blood pressure. Somatic afferent stimulation activates neurons in the hypothalamic PVN. Parvocellular PVN neurons project to sympathoexcitatory cardiovascular regions of the rostral ventrolateral medulla (rVLM). Electroacupuncture (EA) stimulates the median nerve (P5-P6) to modulate sympathoexcitatory responses. We hypothesized that the PVN and its projections to the rVLM participate in the EA-modulation of sympathoexcitatory cardiovascular responses. Cats were anesthetized and ventilated. Heart rate and mean blood pressure were monitored. Application of bradykinin every 10-min on the gallbladder induced consistent pressor reflex responses. Thirty-min of bilateral EA stimulation at acupoints P5-P6 reduced the pressor responses for at least 60-min. Inhibition of the PVN with naloxone reversed the EA-inhibition. Responses of cardiovascular barosensitive rVLM neurons evoked by splanchnic nerve stimulation were reduced by EA and then restored with opioid receptor blockade in the PVN. EA at P5-P6 decreased splanchnic evoked activity of cardiovascular barosensitive PVN neurons that also project directly to the rVLM. PVN neurons labeled with retrograde tracer from rVLM were co-labeled with µ-opioid receptors and juxtaposed to endorphinergic fibers. Thus, the PVN and its projection to rVLM are important in processing acupuncture modulation of elevated blood pressure responses through a PVN opioid mechanism.

Estrogen therapy may counterbalance eutrophic remodeling of coronary arteries and increase bradykinin relaxation in a rat model of menopausal hypertension.

OBJECTIVE: Hypertension causes adverse remodeling and vasomotor alterations in coronaries. Hormones such as estrogen may help counterbalance some of these effects. The aim of this study was to analyze the effects of ovariectomy and estrogen therapy in a rat model of menopausal hypertension induced by angiotensin II (AII). METHODS: We investigated diameter, tone, and mechanics of intramural coronaries taken from ovariectomized female rats (n = 11) that received chronic AII treatment to induce hypertension, and compared the results with those found in female rats that were also given estrogen therapy (n = 11). The "hypertensive control" group (n = 11) underwent an abdominal sham operation, and received AII. After 4 weeks of AII treatment, side branches of left anterior descendent coronary (approximately 200 µm in diameter) were isolated, cannulated with plastic microcannulas at both ends, and studied in vitro in a vessel chamber. The inner and outer diameter of the arteries were measured by microangiometry, and spontenuous tone, wall thickness, wall cross-sectional area, tangential stress, incremental distensibility, circumferential incremental elastic modulus, thromboxane agonist-induced tone, and bradykinin-induced dilation were calculated. RESULTS: In hypertension, intramural small coronaries show inward eutrophic remodeling after ovariectomy comparing with hypertensive controls. Estrogen therapy had an opposite effect on vessel diameter. Hormone therapy led to an increase in spontaneous tone, allowing for greater dilatative capacity. CONCLUSIONS: Estrogen may therefore be considered to counterbalance some of the adverse changes seen in the wall of intramural coronaries in the early stages of chronic hypertension.

Evaluation of Anti-Inflammatory Effects of Helminthostachys zeylanica Extracts via Inhibiting Bradykinin-Induced MMP-9 Expression in Brain Astrocytes.

Phytochemicals present in vegetables, fruits, and herbs are believed to reduce the risk of several major diseases including cardiovascular or neurodegenerative disorders. The roots of the fern Helminthostachys zeylanica (L.) Hook. (Ophioglossaceae) have been used for centuries in the treatment of inflammation and as a folk medicine in several countries. The plant has been shown to possess an array of medicinal properties, including antioxidants and anti-inflammatory activities. Moreover, a rising level of matrix metalloproteinase-9 (MMP-9) has been found in blood fluid of these patients suffering from brain inflammatory diseases, which may be considered an inflammatory biomarker in several inflammatory diseases including the central nervous system (CNS) inflammation. Previously, we have demonstrated the signaling mechanisms of bradykinin (BK)-induced MMP-9 expression in brain astrocytes. Herein, we evaluate the effects of H. zeylanica extracts on BK-induced MMP-9 expression in brain astrocytes and its influencing mechanism. The results showed that H. zeylanica extracts, including E0, E1, and E2 significantly reduce MMP-9 induced by BK in brain astrocytes (RBA-1 cells). These H. zeylanica extracts can inhibit BK-stimulated phosphorylation of c-Src, Pyk2, and PKC(α/δ). Moreover, BK-stimulated NADPH oxidase (Nox)-derived reactive oxygen species (ROS) generation has also been attenuated by pretreatment with these extracts, suggesting that the H. zeylanica extracts have an antioxidative activity. We further demonstrated that the H. zeylanica extracts blocked activation of MAPKs (e.g., ERK1/2 and p38 MAPK) by BK. These data indicated that the H. zeylanica extracts may be has anti-inflammatory activity by reducing BK-induced ROS-dependent MMP-9 expression via these related pathways in brain astrocytes.

Testosterone Replacement Therapy Prevents Alterations of Coronary Vascular Reactivity Caused by Hormone Deficiency Induced by Castration.

The present study aimed to determine the effects of chronic treatment with different doses of testosterone on endothelium-dependent coronary vascular reactivity in male rats. Adult male rats were divided into four experimental groups: control (SHAM), castrated (CAST), castrated and immediately treated subcutaneously with a physiological dose (0.5 mg/kg/day, PHYSIO group) or supraphysiological dose (2.5 mg/kg/day, SUPRA group) of testosterone for 15 days. Systolic blood pressure (SBP) was assessed at the end of treatment through tail plethysmography. After euthanasia, the heart was removed and coronary vascular reactivity was assessed using the Langendorff retrograde perfusion technique. A dose-response curve for bradykinin (BK) was constructed, followed by inhibition with 100 µM L-NAME, 2.8 µM indomethacin (INDO), L-NAME + INDO, or L-NAME + INDO + 0.75 µM clotrimazole (CLOT). We observed significant endothelium-dependent, BK-induced coronary vasodilation, which was abolished in the castrated group and restored in the PHYSIO and SUPRA groups. Furthermore, castration modulated the lipid and hormonal profiles and decreased body weight, and testosterone therapy restored all of these parameters. Our results revealed an increase in SBP in the SUPRA group. In addition, our data led us to conclude that physiological concentrations of testosterone may play a beneficial role in the cardiovascular system by maintaining an environment that is favourable for the activity of an endothelium-dependent vasodilator without increasing SBP.

Hyaluronan modulates TRPV1 channel opening, reducing peripheral nociceptor activity and pain.

Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.

Activation of Mas Oncogene-Related G Protein-Coupled Receptors Inhibits Neurochemical Alterations in the Spinal Dorsal Horn and Dorsal Root Ganglia Associated with Inflammatory Pain in Rats.

Mas oncogene-related G protein-coupled receptor C (MrgC) is unequally expressed in sensory ganglia and has been shown to modulate pathologic pain. This study investigated the mechanism underlying the effect of MrgC receptors on inflammatory pain. Intrathecal administration of the selective MrgC receptor agonist bovine adrenal medulla 8-22 (BAM8-22) (30 nmol) inhibited complete Freund's adjuvant-evoked hyperalgesia. This was associated with the inhibition of protein kinase C-γ and phosphorylated extracellular signal-regulated protein kinase in the spinal cord and/or dorsal root ganglia (DRG). The complete Freund's adjuvant injection in the hindpaw induced an increase in Gq, but not Gi and Gs, protein in the spinal dorsal horn. This increase was inhibited by the intrathecal administration of BAM8-22. The exposure of DRG cultures to bradykinin (10 µM) and prostaglandin E2 (1 µM) increased the expression of calcitonin gene-related peptide (CGRP) and neuronal nitric oxide synthase in small- and medium-sized neurons as well as the levels of CGRP, aspartate, and glutamate in the cultured medium. The bradykinin/prostaglandin E2-induced alterations were absent in the presence of BAM8-22 (10 nM). These results suggest that the activation of MrgC receptors can modulate the increase in the expression of CGRP and neuronal nitric oxide synthase as well as the release of CGRP and excitatory amino acids in DRG associated with inflammatory pain. This modulation results in the inhibition of pain hypersensitivity by suppressing the expression of Gq protein and protein kinase C-γ and extracellular signal-regulated protein kinase signaling pathways in the spinal cord and/or DRG. The present study suggests that MrgC receptors may be a novel target for relieving inflammatory pain.

Serine-727 phosphorylation activates hypothalamic STAT-3 independently from tyrosine-705 phosphorylation.

Transcriptional activity of signal transducer and activator of transcription-3 (STAT-3) is a key element in the central regulation of appetite and energy homeostasis. Activation of hypothalamic STAT-3 has been attributed to cytokine-promoted phosphorylation at tyrosine-705 (Tyr-705). In nonhypothalamic cells, STAT-3 is also phosphorylated at serine-727 (Ser-727), but the functional significance of Ser-727 in the regulation of hypothalamic STAT-3 is not known. We used 2 hypothalamic cell lines and analyzed the effects of various hormones on STAT-3-dependent reporter gene activity and observed that IFN-γ, epidermal growth factor (EGF), and bradykinin (BK) induce similar STAT-3 reporter activation. EGF and BK solely increased Ser-727 and IFN-γ increased Tyr-705 phosphorylation of STAT-3. Specific inhibition of ERK-1/2 activity blocked EGF- and BK-induced STAT-3 activation and Ser-727 phosphorylation. BK-induced ERK-1/2 activation occurred via EGF receptor transactivation. Consequently, the BK-mediated effects on STAT-3 were blocked by a specific EGF receptor antagonist. Next, we analyzed the effects of IFN-γ and EGF on the expression of the STAT-3-dependent genes thyroliberin-releasing hormone and suppressors of cytokine signaling-3. EGF but not IFN-γ enhanced thyroliberin-releasing hormone expression via STAT-3. With regard to suppressors of cytokine signaling-3, we observed prolonged expression induced by IFN-γ and a transient effect of EGF that required coactivation of the activator protein-1. Thus, EGF-promoted Ser-727 phosphorylation by ERK-1/2 is not only sufficient to fully activate hypothalamic STAT-3, but, in terms of targeted genes and required cofactors, entails distinct modes of STAT-3 actions compared with IFN-γ-induced Tyr-705 phosphorylation.

Enteral supplements of a carbon monoxide donor CORM-A1 protect against cerebrovascular dysfunction caused by neonatal seizures.

Cerebral blood flow dysregulation caused by oxidative stress contributes to adverse neurologic outcome of seizures. A carbon monoxide (CO) donor CORM-A1 has antioxidant and cytoprotective properties. We investigated whether enteral supplements of CORM-A1 can improve cerebrovascular outcome of bicuculline-induced seizures in newborn piglets. CORM-A1 (2 mg/kg) was given to piglets via an oral gastric tube 10 minutes before or 20 minutes after seizure onset. Enteral CORM-A1 elevated CO in periarachnoid cerebrospinal fluid and produced a dilation of pial arterioles. Postictal cerebral vascular responses to endothelium-, astrocyte-, and vascular smooth muscle-dependent vasodilators were tested 48 hours after seizures by intravital microscopy. The postictal responses of pial arterioles to bradykinin, glutamate, the AMPA receptor agonist quisqualic acid, ADP, and heme were greatly reduced, suggesting that seizures cause injury to endothelial and astrocyte components of the neurovascular unit. In contrast, in the two groups of piglets receiving enteral CORM-A1, the postictal cerebral vascular responsiveness to these dilators was improved. Overall, enteral supplements of CORM-A1 before or during seizures offer a novel effective therapeutic option to deliver cytoprotective mediator CO to the brain, reduce injury to endothelial and astrocyte components of cerebral blood flow regulation and to improve the cerebrovascular outcome of neonatal seizures.

Medicinas tradicionales andinas y su despenalización: entrevista con Walter Álvarez Quispe / Decriminalizing traditional Andean medicine: an interview with Walter Álvarez Quispe

Walter Álvarez Quispe, terapeuta kallawaya y biomédico especializado en cirugía general y ginecología, presenta la lucha de los terapeutas tradicionales y alternativos por la depenalización de estos sistemas médicos andinos realizada entre 1960 y 1990. Bolivia se torna el primer país en América Latina y el Caribe en despenalizar la medicina tradicional antes de los planteamientos de la Conferencia Internacional sobre Atención Primaria de Salud (Alma-Ata, 1978). Los datos aportados por el entrevistado aseguran que los logros alcanzados, principalmente por los kallawayas, responden a un proyecto propio y autónomo. Estas conquistas no se deben a las políticas oficiales de interculturalidad en salud, aunque busquen atribuirse para sí los logros alcanzados.

Walter Álvarez Quispe, a Kallawaya healer and biomedical practitioner specializing in general surgery and gynecology, presents the struggle of traditional and alternative healers to get their Andean medical systems depenalized between 1960 and 1990. Bolivia was the first country in Latin America and the Caribbean to decriminalize traditional medicine before the proposals of the International Conference on Primary Health Care (Alma-Ata, 1978). The data provided by the interviewee show that the successes achieved, mainly by the Kallawayas, stem from their own independent initiative. These victories are not the result of official policies of interculturality in healthcare, although the successes achieved tend to be ascribed to them.

Effect of maternal nutrient restriction and melatonin supplementation from mid to late gestation on vascular reactivity of maternal and fetal placental arteries.

INTRODUCTION: Maternal nutrient restriction and decreased scotophase concentrations of melatonin have been associated with severely compromised pregnancies. We hypothesized that melatonin supplementation in a compromised pregnancy enhances the bradykinin (BK)-induced relaxations of placental arteries thereby ensuring sufficient umbilical blood flow to the developing fetus. METHODS: Pregnant ewes (n = 31) were fed an adequate (ADQ) or nutrient restricted (RES) diet supplemented with 5 mg of melatonin (MEL) or without melatonin (CON) from day 50 to 130 of gestation. On day 130 of gestation, the maternal (caruncular; CAR) and fetal (cotyledonary; COT) placental arteries were suspended in organ chambers for isometric tension recording. RESULTS: There were no treatment or dietary effects on CAR arteries for any vasoactive agent. However, in COT arteries, MEL ewes were more sensitive (P < 0.01) to bradykinin-induced relaxation than CON ewes. There was a melatonin by nutritional level interaction (P < 0.01) with sodium nitroprusside-induced relaxation of COT arteries where CON-RES were more sensitive to sodium nitroprusside compared to CON-ADQ, which was in contrast to when ewes were fed MEL. There was a significant melatonin by nutritional interaction (P = 0.04) for responsiveness to norepinephrine. The sensitivity of the COT arteries to norepinephrine in CON-RES ewes was decreased compared to CON-ADQ. Melatonin supplementation, regardless of maternal dietary intake, resulted in COT arteries having similar responsiveness to CON-RES ewes. CONCLUSION: An increase in placental vessel sensitivity to bradykinin-induced relaxation may contribute to melatonin-induced increases in umbilical artery blood flow.