Injection Of Bradykinin Research Articles

CU06-1004 alleviates vascular hyperpermeability in a murine model of hereditary angioedema by protecting the endothelium.

Over-release of the vasoactive peptide bradykinin (BK) due to mutation in the SERPING1 gene is the leading cause of hereditary angioedema (HAE). BK directly activates endothelial cells and increases vascular permeability by disrupting the endothelial barrier, leading to angioedema affecting face, lips, extremities, gastrointestinal tract, and larynx. Although various pharmacological treatment options for HAE became available during the last decade, they are presently limited and pose a major economic burden on patients. To identify additional therapeutic options for HAE, we evaluated the effect of CU06-1004, an endothelial dysfunction blocker, on BK-induced vascular hyperpermeability and the HAE murine model. To investigate the effect of CU06-1004 on BK-induced vascular hyperpermeability in vivo, we pre-administrated WT mice with the drug and then induced vascular leakage through intravenous injection of BK and observed vascular alternation. Then, SERPING1 deficient mice were used for a HAE murine model. For an in vitro model, the HUVEC monolayer was pre-treated with CU06-1004 and then stimulated with BK. Bradykinin disrupted the endothelial barrier and formed interendothelial cell gaps, leading to hyperpermeability in vivo and in vitro. However, CU06-1004 treatment protected the endothelial barrier by suppressing Src and myosin light chain activation via BK and alleviated hyperpermeability. Our study shows that CU06-1004 oral administration significantly reduced vascular hyperpermeability in the HAE murine model by protecting the endothelial barrier function against BK stimulation. Therefore, protecting endothelium against BK with CU06-1004 could serve as a potential prophylactic/therapeutic approach for HAE patients.

Hydroethanolic Leaf Extract of Cordia vignei Hutch and Dalziel Inhibits Carrageenan-Induced Foot Oedema in Chicks, Prostaglandin E2-Induced Paw Oedema in Mice, and Bradykinin-Induced Paw Oedema in Mice.

Cordia vignei Hutch and Dalziel (Fam. Boraginaceae) is a woody plant found in west tropical Africa. The aim of this research is to find out if the leaf extract of this plant prevents oedema in animal models. (a) Inflammation was induced in the animals by injecting 100 µl of 2% lambda carrageenan into the subplantar tissue of the right footpads of 7-day-old chicks 1 h before or after oral administration of 30-300 mgkg-1 CVE. Oedema was measured for 5 h using the water displacement method. (b) Oedema was induced in ICR mice by subplantar injection of prostaglandin E2 (PGE2) (50 µl of 1 nM) 30 minutes before or after CVE administration. Oedema was measured for 3 h. (c) Oedema was induced in ICR mice by subplantar injection of bradykinin (BK) (10 nmol/paw) 30 min before or after administration of extract. We found that CVE significantly (P < 0.05) prevented inflammation that was induced by injecting carrageenan into the footpads of the chicks. Also, we observed that CVE prevented inflammation produced by injecting PGE2 into the subplantar tissue of mice. Finally, we also report that CVE prevented inflammation produced by injecting BK into the subplantar tissues of mice. All these effects were observed in both preventive and curative protocols. We conclude that Cordia vignei leaf extract has potential anti-inflammatory activity.

Bradykinin 2 receptors contribute to the exaggerated exercise pressor reflex in a rat model of simulated peripheral artery disease.

We investigated the role played by bradykinin 2 (B2) receptors in the exaggerated exercise pressor reflex in rats with a femoral artery ligated for 72 h to induce simulated peripheral artery disease (PAD). We hypothesized that in decerebrate, unanesthetized rats with a ligated femoral artery, hindlimb arterial injection of HOE-140 (100 ng, B2 receptor antagonist) would reduce the pressor response to 30 s of electrically induced 1 Hz hindlimb skeletal muscle contraction, and 30 s of 1 Hz hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). We hypothesized no effect of HOE-140 in sham-operated "freely perfused" rats. In both freely perfused (n = 4) and "ligated" (n = 4) rats, we first confirmed efficacious B2 receptor blockade by demonstrating that HOE-140 injection significantly reduced (P < 0.05) the peak increase in mean arterial pressure (peak ΔMAP) in response to hindlimb arterial injection of bradykinin. In subsequent experiments, we found that HOE-140 reduced the peak ΔMAP response to muscle contraction in ligated (n = 14; control: 23 ± 2; HOE-140: 17 ± 2 mmHg; P = 0.03) but not freely perfused rats (n = 7; control: 17 ± 3; HOE-140: 18 ± 4 mmHg; P = 0.65). Furthermore, HOE-140 had no effect on the peak ΔMAP response to stretch in ligated rats (n = 14; control: 37 ± 4; HOE-140: 32 ± 5 mmHg; P = 0.13) but reduced the integrated area under the blood pressure signal over the final ∼20 s of the maneuver. The data suggest that B2 receptors contribute to the exaggerated exercise pressor reflex in rats with simulated PAD, and that contribution includes a modest role in the chronic sensitization of the mechanically activated channels/afferents that underlie mechanoreflex activation.

Age-related changes in peripheral nociceptor function

Pain and pain management in the elderly population is a significant social and medical problem. Pain sensation is a complex phenomenon that typically involves activation of peripheral pain-sensing neurons (nociceptors) which send signals to the spinal cord and brain that are interpreted as pain, an unpleasant sensory experience. In this work, young (4–5 months) and aged (26–27 months) Fischer 344 x Brown Norway (F344xBN) rats were examined for nociceptor sensitivity to activation by thermal (cold and heat) and mechanical stimulation following treatment with inflammatory mediators and activators of transient receptor potential (TRP) channels. Unlike other senses that decrease in sensitivity with age, sensitivity of hindpaw nociceptors to thermal and mechanical stimulation was not different between young and aged F344xBN rats. Intraplantar injection of bradykinin (BK) produced greater thermal and mechanical allodynia in aged versus young rats, whereas only mechanical allodynia was greater in aged rats following injection of prostaglandin E2 (PGE2). Intraplantar injection of TRP channel activators, capsaicin (TRPV1), mustard oil (TRPA1) and menthol (TRPM8) each resulted in greater mechanical allodynia in aged versus young rats and capsaicin-induced heat allodynia was also greater in aged rats. A treatment-induced allodynia that was greater in young rats was never observed. The anti-allodynic effects of intraplantar injection of kappa and delta opioid receptor agonists, salvinorin-A and D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, were greater in aged than young rats, whereas mu opioid receptor agonists, [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) and morphine, were not effective in aged rats. Consistent with these observations, in primary cultures of peripheral sensory neurons, inhibition of cAMP signaling in response to delta and kappa receptor agonists was greater in cultures derived from aged rats. By contrast, mu receptor agonists did not inhibit cAMP signaling in aged rats. Thus, age-related changes in nociceptors generally favor increased pain signaling in aged versus young rats, suggesting that changes in nociceptor sensitivity may play a role in the increased incidence of pain in the elderly population. These results also suggest that development of peripherally-restricted kappa or delta opioid receptor agonists may provide safer and effective pain relief for the elderly.

Venoconstrictor responses to activation of bradykinin-sensitive pericardial afferents involve the region of the hypothalamic paraventricular nucleus.

Veins are important in the control of venous return, cardiac output, and cardiovascular homeostasis. However, the effector systems modulating venous function remain to be fully elucidated. We demonstrated that activation of bradykinin‐sensitive pericardial afferents elicited systemic venoconstriction. The hypothalamic paraventricular nucleus (PVN) is an important site modulating autonomic outflow to the venous compartment. We tested the hypothesis that the PVN region is involved in the venoconstrictor response to pericardial injection of bradykinin. Rats were anesthetized with urethane/alpha chloralose and instrumented for recording arterial pressure, vena caval pressure, and mean circulatory filling pressure (MCFP), an index of venous tone. The rats were fitted with a pericardial catheter and PVN injector guide tubes. Mean arterial pressure (MAP), heart rate (HR), and MCFP responses to pericardial injection of bradykinin (1, 10 µg/kg) were recorded before and after PVN injection of omega conotoxin GVIA (200 ng/200 nl). Pericardial injection of saline produced no systematic effects on MAP, HR, or MCFP. In contrast, pericardial injection of bradykinin was associated with short latency increases in MAP (16 ± 4 to 18 ± 2 mm Hg) and MCFP 0.35 ± 0.19 to 1.01 ± 0.27 mm Hg. Heart rate responses to pericardial BK were highly variable, but HR was significantly increased (15 ± 9 bpm) at the higher BK dose. Conotoxin injection in the PVN region did not affect baseline values for these variables. However, injection of conotoxin into the area of the PVN largely attenuated the pressor (−1 ± 3 to 6 ± 3 mm Hg), MCFP (−0.19 ± 0.07 to 0.20 ± 0.18 mm Hg), and HR (4 ± 14 bpm) responses to pericardial bradykinin injection. We conclude that the PVN region is involved in the venoconstrictor responses to pericardial bradykinin injection.

Hemodynamic patterns associated with activation of bradykinin-sensitive pericardial afferents.

The heart is endowed with reflexogenic areas capable of powerful blood pressure responses. Relatively little work has studied the hemodynamic mechanisms underlying these responses and whether these are sexually dimorphic. We hypothesized that activation of bradykinin-sensitive pericardial afferents would produce a sexually dimorphic cardiac output response. Male and female Sprague Dawley rats were anesthetized and instrumented with catheters for recording arterial pressure, with an aortic arch flow probe to record cardiac output and with a catheter in the pericardial sac. Mean arterial pressure (MAP), cardiac index (CI) and total peripheral resistance index (TPRI) responses to pericardial bradykinin injection (0.1, 1 μg/kg) were recorded. Pericardial bradykinin injection caused similar increases in MAP in male and female rats. However, the underlying hemodynamic patterns varied considerably. We identified a cluster of CI responders and TPRI responders in both male and female rats. Within CI responders, females exhibited greater CI increases than males. Conversely, in TPRI responders, males exhibited a greater TPRI increase than females. We conclude that aggregate activation of bradykinin-sensitive pericardial afferents is associated with a relatively uniform pressor response but different hemodynamic patterns with males exhibiting a more robust vascular response and females a more robust cardiac output response.

Analgesic Effect of Ziziphus abyssinica Involves Inhibition of Inflammatory Mediators and Modulation of KATP Channels, Opioidergic and Nitrergic Pathways.

The diversity offered by natural products has timelessly positioned them as a good source for novel therapeutics for the management of diverse medical conditions, including pain. This study evaluated hydro-ethanolic root bark extract of Ziziphus abyssinica (ZAE) as well as β-amyrin and polpunonic acid isolated from the plant for analgesic property. The study also investigated the mechanism responsible for this action in the extract. The antinociceptive potential of ZAE (30, 100, and 300 mg/kg, p. o.) was assessed using the tail-immersion test (TIT), acetic acid-induced writhing test (AAT), and formalin test (FT). The extract’s effect on acute and chronic musculoskeletal pain was also assessed by administering carrageenan unilaterally into the rat gastrocnemius muscles and measuring pain at 12 h and 10 days for acute and chronic pain respectively. The involvement of pro-inflammatory mediators (prostaglandin E2, bradykinin, TNF-α, and IL-1β) was assessed. The possible pathways mediating the observed analgesic effect of ZAE were further assessed using the antagonists: naloxone, glibenclamide, NG-L-nitro-arginine methyl ester (L-NAME), atropine, nifedipine, and yohimbine in the FT. Also the analgesic effect of two triterpenoid compounds, β-amyrin and polpunonic acid, previously isolated from the plant was assessed using the TIT. The anti-nociceptive activity of ZAE was demonstrated in the TIT by the significant (p < 0.05) increase in tail withdrawal threshold in ZAE-treated mice. ZAE also markedly reduced writhing and paw licking responses in both AAT and FT and significantly (p < 0.05) attenuated both acute and chronic musculoskeletal pain. ZAE also significantly reversed hyperalgesia induced by intraplantar injection of PGE2, bradykinin, TNF-α, and IL-1β. Furthermore, data revealed the involvement of opioidergic, ATP-sensitive K+ channels and NO-cGMP pathways in the analgesic effect of ZAE. Both β-amyrin and polpunonic acid exhibited analgesic activity in the tail suspension test. Our study demonstrates ZAE as an important source of new therapeutic agents for pain management.

Junctophilin-4 facilitates inflammatory signalling at plasma membrane-endoplasmic reticulum junctions in sensory neurons.

Rat somatosensory neurons express a junctional protein, junctophilin-4 (JPH4) JPH4 is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the junctions between plasma membrane and endoplasmic reticulum in these neurons. Knockdown of JPH4 impairs endoplasmic reticulum Ca2+ store refill and junctional Ca2+ signalling in sensory neurons. In vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly attenuated experimentally induced inflammatory pain in rats. Junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms. Junctions of endoplasmic reticulum and plasma membrane (ER-PM junctions) form signalling nanodomains in eukaryotic cells. ER-PM junctions are present in peripheral sensory neurons and are important for the fidelity of G protein coupled receptor (GPCR) signalling. Yet little is known about the assembly, maintenance and physiological role of these junctions in somatosensory transduction. Using fluorescence imaging, proximity ligation, super-resolution microscopy, in vitro and in vivo gene knockdown we demonstrate that a member of the junctophilin protein family, junctophilin-4 (JPH4), is necessary for the formation of store operated Ca2+ entry (SOCE) complex at the ER-PM junctions in rat somatosensory neurons. Thus we show that JPH4 localises to the ER-PM junctional areas and co-clusters with SOCE proteins STIM1 and Orai1 upon ER Ca2+ store depletion. Knockdown of JPH4 impairs SOCE and ER Ca2+ store refill in sensory neurons. Furthermore, we demonstrate a key role of the JPH4 and junctional nanodomain Ca2+ signalling in the pain-like response induced by the inflammatory mediator bradykinin. Indeed, an in vivo knockdown of JPH4 in the dorsal root ganglion (DRG) sensory neurons significantly shortened the duration of nocifensive behaviour induced by hindpaw injection of bradykinin in rats. Since the ER supplies Ca2+ for the excitatory action of multiple inflammatory mediators, we suggest that junctional nanodomain Ca2+ signalling maintained by JPH4 is an important contributor to the inflammatory pain mechanisms.

Intra-arterial injection of bradykinin produces reflex cardiorespiratory changes involving histamine receptors in anesthetized rats

Objectives: It is well known that intra-arterial injection of nociceptive agent produces vasosensory reflex responses altering cardiorespiratory parameters. The role of various inflammatory mediators is also implicated in the regulation of these reflex responses. However, the role of histamine in this regard is not clear. This study was performed to understand the role of H1 and H2 receptors in modulating the cardiorespiratory responses evoked after i.a. injection of bradykinin (BK). Materials and Methods: Male albino rats were anesthetized with an intra-peritoneal injection of urethane (1.5 g/kg). Tracheostomy was performed to keep the respiratory tract patent. The femoral artery was cannulated proximally by pediatric i.v. cannula (24 G, double ported). This cannulation was used for the blood pressure (BP) recording as well as for the drugs instillation as it contains double port with injection valve. The effect of BK (1 µM) on BP, electrocardiographic, and respiration was recorded for 30 min. The respiratory frequency, respiratory minute volume, mean arterial pressure, and heart rate were computed from the original tracings and the data were presented as mean ± SEM. Results: Intra-arterial injection of BK produced immediate hyperventilatory (50% from initial), hypotensive (40% from initial), and bradycardiac responses (17% from initial) of shorter latency (5–8 s) indicating the neural mechanisms in producing the responses. Pre-treatment with pheniramine maleate significantly attenuated the BK-induced hyperventilatory (11% from initial), hypotensive (8% from initial), and bradycardiac responses (2% from initial). Conclusion: Our data provide evidences for the involvement of H1 and H2 receptors in producing the BK-induced vasosensory reflex responses modulating the cardiovascular parameters in anesthetized rats.

Activation of Bradykinin‐Sensitive Pericardial Afferents Increases Splanchnic Nerve Activity

Sympathetic drive plays a key role in control of cardiovascular function. The heart performs an important sensory function via a variety of cardiac receptors that modulate sympathetic drive. Bradykinin‐sensitive sympathetic afferent nerves from the heart can trigger marked increases in efferent sympathetic nerve activity to some cardiovascular targets such as the kidney. We reported previously that pericardial injection of bradykinin increased venous tone and cardiac output. The splanchnic vascular compartment plays an important role in control of venous return and cardiac output. This work tested the hypothesis that activation of bradykinin‐sensitive pericardial afferents increases postganglionic splanchnic sympathetic nerve activity.Sprague Dawley rats were anesthetized with a cocktail of alpha chloralose (80 mg/kg) and urethane (800 mg/kg). Catheters were placed in the femoral artery and vein to record arterial pressure (MAP) and heart rate (HR). A PE10 catheter was placed in the pericardial space for the injection of bradykinin (BK), an agent known to stimulate cardiac sympathetic afferents. The chest was closed and the rat allowed to breathe spontaneously. The splanchnic sympathetic nerve was isolated via a dorsal approach and placed on recording electrodes. After stabilization, the blood pressure, heart rate, and integrated splanchnic nerve activity (SPLNA) responses to pericardial injection of BK or vehicle were recorded. Responses were also recorded for intravenous injection of dimethylphenylpiperazinium (DMPP 150 ug/kg), a ganglionic stimulant, and a ganglion blocker (hexamethonium 20 mg/kg).Pericardial injection of saline vehicle had little effect (MAP: 0.1±0.4 mm Hg; HR: 1.2±1.4 bpm; SPLNA 1±2%). In contrast, intravenous injection of a ganglionic stimulant, DMPP, increased MAP (23±1 mm Hg) and SPLNA (95±46%). Pericardial injection of BK (10 ug/kg) increased MAP (21±3 mm Hg), HR (28±7 bpm) and SPLNA (93±37%). Ganglionic blockade with hexamethonium reduced SPLNA (−75±6%) and abolished the responses to pericardial injection of BK (MAP: −1±3 mm Hg; HR: −2±1 bpm; SPLNA: 7±30 %).We interpret these data to indicate that activation of bradykinin‐sensitive pericardial afferents increases postganglionic splanchnic sympathetic nerve activity. This finding is consistent with the view that activation of the cardiac sympathetic afferent reflex elicits a venoconstrictor response to increase venous return from the splanchnic vascular compartment.Support or Funding InformationSupported by NIH R01 HL136741‐03 (DM), R01 DK114663‐1 (HZ), and the Basic Biomedical Sciences program of the Sanford School of Medicine, University of South Dakota.

Changes in Splanchnic Hemodynamics Contribute to the Cardiovascular Responses Following Activation of Bradykinin‐Sensitive Pericardial Afferents

Sympathetic drive is a major controller of cardiovascular function. The heart performs an important sensory function via a variety of cardiac receptors that modulate cardiovascular responses by both neural and humoral reflexes. Bradykinin‐sensitive sympathetic afferent nerves from the heart can trigger marked increases in efferent sympathetic nerve activity to some cardiovascular target organs such as the kidney, arteries and veins. We reported previously that pericardial injection of bradykinin increased venous tone and cardiac output. The splanchnic vascular compartment plays an important role in control of venous return and cardiac output. This work tested the hypothesis that activation of bradykinin‐sensitive pericardial afferents modulates splanchnic hemodynamics.Sprague Dawley rats were anesthetized with a cocktail of alpha chloralose (80 mg/kg) and urethane (800 mg/kg). Catheters were placed in the femoral artery and vein to record arterial pressure (MAP) and heart rate (HR). A PE10 catheter was placed in the pericardial space for the injection of bradykinin (BK), an agent known to stimulate cardiac sympathetic afferents. The chest was closed and the rats allowed to breathe spontaneously. The portal vein and mesenteric artery were exposed via a midline laparotomy and fitted with transit time flow probes. After stabilization, the blood pressure, heart rate, portal venous flow (PF) and mesenteric flow (MF) responses to pericardial injection of BK or vehicle were recorded.Pericardial injection of saline vehicle had little effect (MAP: 2±2 mm Hg; HR: 13±6 bpm; PF: −5±1%; MF: −3±1%). In contrast, pericardial injection of BK (10 ug/kg) increased MAP (16±3 mm Hg) and HR (28±5 bpm). After an initial transient decrease, PF (10±2%) and MF (5±2%) were increased during the plateau phase of the MAP response (60–120 seconds).We interpret these data to indicate that activation of bradykinin‐sensitive pericardial afferents increases portal venous flow. This finding is consistent with the view that activation of the cardiac sympathetic afferent reflex elicits a venoconstrictor response to increase venous return from the splanchnic compartment.Support or Funding InformationSupported by NIH R01 HL136741‐03, and the Basic Biomedical Sciences program of the Sanford School of Medicine.

Instillation of bradykinin into femoral artery elicits cardiorespiratory reflexes involving perivascular afferents in anesthetized rats.

The physiology of baroreceptors and chemoreceptors present in large blood vessels of the heart is well known in the regulation of cardiorespiratory functions. Since large blood vessels and peripheral blood vessels are of the same mesodermal origin, therefore, involvement of the latter in the regulation of cardiorespiratory system is expected. The role of perivascular nerves in mediating cardiorespiratory alterations produced after intra-arterial injection of a nociceptive agent (bradykinin) was examined in urethane-anesthetized male rats. Respiratory frequency, blood pressure, and heart rate were recorded for 30 min after the retrograde injection of bradykinin/saline into the femoral artery. In addition, paw edema was determined and water content was expressed as percentage of wet weight. Injection of bradykinin produced immediate tachypneic, hypotensive and bradycardiac responses of shorter latency (5-8 s) favoring the neural mechanisms involved in it. Injection of equi-volume of saline did not produce any responses and served as time-matched control. Paw edema was observed in the ipsilateral hind limb. Pretreatment with diclofenac sodium significantly attenuated the bradykinin-induced responses and also blocked the paw edema. Ipsilateral femoral and sciatic nerve sectioning attenuated bradykinin-induced responses significantly, indicating the origin of responses from the local vascular bed. Administration of bradykinin in the segment of an artery produced reflex cardiorespiratory changes by stimulating the perivascular nociceptors involving prostaglandins. This is a novel study exhibiting the role of peripheral blood vessels in the regulation of the cardiorespiratory system.

Activation of bradykinin-sensitive pericardial afferents increases systemic venous tone in conscious rats.

Our understanding of reflex regulation of veins lags behind that of the arterial system. While the cardiac sympathetic afferent reflex (CSAR) exerts control over sympathetic outflow, its effect on venous tone is not known. We tested the hypothesis that activation of pericardial bradykinin sensitive afferents elicits systemic venoconstriction. Male and female Sprague Dawley rats were chronically instrumented for measurement of arterial pressure and mean circulatory filling pressure, an index of venous tone, and with an indwelling pericardial catheter. Mean arterial pressure, heart rate and mean circulatory filling pressure responses were assessed in conscious rats in response to graded pericardial injections of bradykinin (1.5-20μg/kg) before and after ganglionic blockade, and to intravenous norepinephrine (0.05-0.8μg/kg). Bradykinin B2 receptor was assessed by Western blot. Pericardial bradykinin injections caused graded increases in mean arterial pressure, heart rate and mean circulatory filling pressure. These responses were markedly attenuated after autonomic blockade. The increments in mean circulatory filling pressure were attenuated in female rats. There were no differences in the venoconstrictor responses to norepinephrine or ventricular bradykinin receptor expression between male and females. We interpret these findings to indicate that activation of bradykinin sensitive pericardial afferents elicits a sexually dimorphic, autonomically mediated systemic venoconstrictor response. Differences in venous smooth muscle responses to norepinephrine or ventricular bradykinin receptor expression do not account for the sexual dimorphism. We conclude that systemic venoconstriction contributes to the overall hemodynamic response to activation of the cardiac sympathetic afferent reflex and that this effect is sexually dimorphic.

Cyclooxygenase activity in bradykinin-induced dermal extravasation. A study in mice and humans

BackgroundNon-allergic angioedema is largely driven by increased plasma levels of bradykinin and over-activation of bradykinin receptor type II (B2), but the specific downstream signalling pathways remain unclear. The aim of this study was to identify signal transduction events involved in bradykinin-induced dermal extravasation. MethodsQuantification of dermal extravasation was accomplished following intradermal (i.d.) injection of bradykinin or the B2 agonist labradimil in mice with endothelial NO-synthase (eNOS) deficiency and in C57BL/6J mice pre-treated with vehicle, NO-synthase or cyclooxygenase (COX) inhibitors. In the multicentre clinical study ABRASE, 38 healthy volunteers received i.d. bradykinin injections into the ventral forearm before and after oral treatment with the COX inhibitor ibuprofen (600 mg). The primary endpoint of ABRASE was the mean time to complete resolution of wheals (TTCR) and the secondary endpoint was the change of maximal wheal size. ResultsNeither NOS inhibitors nor eNOS deficiency altered bradykinin-induced extravasation. In striking contrast, the COX inhibitors ibuprofen, diclofenac, SC560 and celecoxib significantly diminished this extravasation when given before injection. As for diclofenac, a similar but significantly lower effect was observed when given after i.d. injection of bradykinin. Similar results were obtained when bradykinin was replaced by labradimil. In volunteers, ibuprofen significantly reduced TTCR (P < 0.001) and maximal wheal size (P = 0.0044). ConclusionThese data suggest that COX activity contributes to bradykinin-induced dermal extravasation in mice and humans. In addition, our findings may open new treatment options and point to a potential activity of drugs interfering with the release of the COX substrate arachidonic acid, e.g. glucocorticoids.

Blocking the transient receptor potential vanilloid-1 does not reduce the exercise pressor reflex in healthy rats.

Controversy exists regarding the role played by transient receptor potential vanilloid-1 (TRPV1) in evoking the exercise pressor reflex. Here, we determine the role played by TRPV1 in evoking this reflex while assessing possible confounding factors arising from TRPV1 antagonists or from the vehicle in which they were dissolved. The exercise pressor reflex was evoked in decerebrated, anesthetized Sprague-Dawley rats by electrical stimulation of the tibial nerve to contract the triceps surae muscles statically. This procedure was repeated before and after injection of the TRPV1 blockers: capsazepine (100 μg/100 μL), ruthenium red (100 μg/100 μL), or iodoresiniferatoxin (IRTX; 1 μg/100 μL). We found that capsazepine decreased the exercise pressor reflex when the drug was dissolved in DMSO (-10 ± 9 mmHg; P = 0.015; n = 7). However, similar reduction was found when DMSO alone was injected (-8 ± 5 mmHg; P = 0.023; n = 5). Capsazepine, dissolved in ethanol (2 ± 6 mmHg; P = 0.49; n = 7), ruthenium red (-4 ± 12 mmHg; P = 0.41; n = 7), or IRTX (4 ± 18 mmHg; P = 0.56; n = 7), did not significantly decrease the exercise pressor reflex. In addition, we found that capsazepine and ruthenium red had "off-target" effects. Capsazepine decreased the pressor response evoked by intra-arterial injection of bradykinin (500 ng/kg; -12 ± 13 mmHg; P = 0.028; n = 9) and α-β-methylene ATP (10 μg/kg; -7 ± 8 mmHg; P = 0.019; n = 10), whereas ruthenium red decreased the ability of the muscle to produce and sustain force (-99 ± 83 g; P = 0.020; n = 7). Our data therefore suggest that TRPV1 does not play a role in evoking the exercise pressor reflex. Additionally, given their strong off-target effects, capsazepine and ruthenium red should not be used for studying the role played by TRPV1 in evoking the exercise pressor reflex.

Retinal proteome associated with bradykinin-induced edema

The plasma kallikrein-stimulated generation of bradykinin (BK) has been implicated in diabetic macular edema (DME). This study characterizes the effects of BK on the ultrastructure and proteome of the rat retina. The effects of intravitreal injection of BK on retinal thickness and vascular ultrastructure in Sprague Dawley rats were analyzed and compared with the effects of VEGF using spectral-domain optical coherence tomography. At 24 h post intravitreal injection of BK or saline vehicle retina were harvested and solubilized proteins were analyzed by mass spectrometry-based proteomics. Proteins were identified using X!Tandem and spectral counts were used as a semiquantitative measurement of protein abundance. Proteins identified from retinal extracts were annotated by Gene Ontology (GO) slim terms and compared with a human DME vitreous proteome. Intravitreal injection of BK and VEGF induced transient increases in retinal thickness of 46 μm (24.6%, p = 0.015) and 39 μm (20.3%, p = 0.004), respectively at 24 h, which were resolved to baseline thicknesses at 96 h post injection. BK and VEGF also increased retinal vessel diameters and tortuosity at 24 h post intravitreal injection. Proteomic analyses identified 1757 non-redundant proteins in the rat retina, including 1739 and 1725 proteins from BK- and saline control-injected eyes, respectively. Eighteen proteins, including two proteins associated with intercellular junctions, filamin A and actinin alpha 4, were decreased by at least 50% (p < 0.05) in retina from BK-injected eyes compare with retina from eyes injected with saline. In addition, 32 proteins were increased by > 2-fold (p < 0.05) in retina from BK-injected eyes. Eight proteins, including complement C3, were identified to be increased in both BK-stimulated rat retina and in human DME vitreous. Western blot analysis showed that Complement 3 levels in vitreous from BK-injected eyes in rats and clinical DME samples were increased by 6.6-fold (p = 0.039) and 4.3-fold (p = 0.02), compared with their respective controls. In summary, this study identifies protein changes in rat retina that are associated with BK-induced retinal thickening, including 8 proteins that were previously reported to be increased in the human DME vitreous proteome.

Capsazepine decrease the pressor response to stimuli other than transient receptor potential vanilloid‐1 agonists

Capsaicin‐induced stimulation of Transient Receptor Potential Vanilloid‐1 (TRPV1) receptors on thin fiber muscle afferents reflexively increases arterial blood pressure in multiple species of mammals. In rats, blocking these receptors with capsazepine reduced the pressor response to contraction of the hindlimb muscles, a finding which suggests that TRPV1 receptors mediate the exercise pressor reflex. Capsazepine, however, has been shown in vitro to block voltage gated calcium channels, raising the possibility that it might have reduced the exercise pressor reflex by another mechanism. To test that alternative, we examined the effect of capsazepine on the pressor responses to intra‐arterial injection of bradykinin and diprotonated phosphate, metabolic by‐products of contraction believed to play a role in the elicitation of the exercise pressor reflex. In decerebrated Sprague‐Dawley rats, we injected bradykinin (1.5 μg/kg, 0.1 mL; n = 9) or diprotonated phosphate (86 mM, pH = 6.0, 0.2 mL; n = 3) into the arterial supply of the hindlimb before and after injecting capsazepine (100 μg/mL, 0.1 mL). We challenged the effectiveness of capsazepine to inhibit TRPV1 receptors by injecting the specific TRPV1 agonist, capsaicin (0.5 μg/kg, 0.1 mL). Capsazepine decreased the pressor response to capsaicin (50 ± 11 mmHg vs.32 ± 14 mmHg [mean ± SD]; p = 0.06, d = 1.03) and to bradykinin (25 ± 8 mmHg vs.13 ± 12 mmHg; p = 0.03, d = 0.85). The effect of capsazepine on the pressor response to diprotonated phosphate was unclear due to a small sample size (44 ± 25 mmHg vs. 31 ± 29 mmHg). Intra‐arterial injection of the vehicle for capsazepine had no effect on the pressor responses to bradykinin, diprotonated phosphate and capsaicin. Our data suggest that capsazepine is not a selective antagonist to TRPV1 receptors and should not be used to test the hypothesis that TRPV1 receptors on thin fiber afferents play a role in evoking the exercise pressor reflex.Support or Funding InformationSupported by NIH P01 HL 134609.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microinjection of endomorphin-1 in the ventrolateral periaqueductal gray induces descending inhibition of cardiac nociception by activating μ-opioid receptor in rats

To observe descending inhibition of cardiac nociception induced by microinjection of endomorphin-1 (EM1) in the ventrolateral periaqueductal gray (VLPAG) in rats effect and explore the role of μ-opioid receptor in mediating this effect. Male SD rats were randomized into electromyography (EMG) group and c-Fos group, both of which were further divided into 5 subgroups, namely 0.9% NaCl group, bradykinin (BK) group, BK+EM1 group, BK+CTOP group, and BK+CTOP+EM1 group. Rat models of cardiac nociception were established by intrapericardial injection of BK. The changes of cardiaosomatic motor reflex induced by BK were observed by assessing EMG responses of the dorsal spinotrapezius muscle; c-Fos expression in the spinal dorsal horn at levels T3-T5 was tested. Compared with 0.9% NaCl, intrapericardial BK injection induced obvious EMG activities and significantly increased c-Fos expression in the spinal dorsal horn at T3-T5 (P &lt; 0.05). Compared with BK injection, microinjection of EM1 in the VLPAG dose-dependently inhibited EMG activities and significantly decreased c-Fos expression (P &lt; 0.05); microinjection of CTOP in the VLPAG produced no significant effect on EMG or c-Fos expression (P &gt; 0.05). Microinjection of CTOP obviously reversed EM1-induced inhibition of EMG activities and c-Fos expression (P &lt; 0.05). Microinjection of EM1 in the VLPAG produces descending inhibition of cardiac nociception in rats by activating μ-opioid receptor.

Analgesic Effects Elicited by Neuroactive Mediators Injected into the ST 36 Acupuncture Point on Inflammatory and Neuropathic Pain in Mice

The present study evaluates whether the injection of serotonin, acetylcholine, glutamate, bradykinin, histamine, or substance P (SP) into the Zusanli (Stomach 36, ST 36) acupoint can also produce the acupuncture-induced antinociceptive effect on inflammatory or neuropathic pain. In this in vivo experimental study, a total of 450 male Swiss mice were used. Mice were injected with saline or complete Freund's adjuvant (CFA) or subjected to sham or chronic constriction injury (CCI) surgery. After the establishment of the inflammatory (4 hours) or the neuropathic pain (3 days), the animals (n = 6) received manual acupuncture, sham acupuncture, or injection of saline, serotonin, acetylcholine, glutamate, bradykinin, histamine, or SP into the ST 36 and were evaluated for up to 24 hours. Mechanical threshold was evaluated, and the L4-L6 dorsal root ganglion was used for analysis of the transient receptor potential vanilloid type 1 overexpression. The mice from both the CFA and CCI models treated with manual acupuncture had significant increases in the thresholds for more than 24 hours. Sham acupuncture stimulation did not change the thresholds. In the mice injected with each of the mediators, the thresholds were significantly increased for all times in both the CFA and CCI models. Transient receptor potential vanilloid type 1 overexpression in CFA and CCI mice was reduced at all times by injection of serotonin, acetylcholine, or SP but not by injection of glutamate, histamine, or bradykinin. Our data suggest that the neuroactive mediators released by acupuncture-induced tissue injury may contribute to acupuncture-induced analgesia.

Activation of Bradykinin‐Sensitive Pericardial Afferents Increases Systemic Venous Tone and Cardiac Output

The venous system plays a critical role in blood pressure regulation by controlling venous return, cardiac filling, and cardiac output. Sympathetic drive is a major controller of venous function. However, outside of the arterial baroreflex, the mechanisms that regulate sympathetic drive to veins remain poorly defined in both physiological and disease conditions. The heart itself has an important sensory function via a variety of cardiac receptors that modulate cardiovascular function by neural and humoral reflexes. Bradykinin‐sensitive sympathetic afferent nerves from the heart can trigger marked increases in efferent sympathetic nerve activity to some cardiovascular target organs such as the kidney. This work tested the hypothesis that the activation of bradykinin‐sensitive cardiac afferents increases venous tone and cardiac output.Sprague Dawley rats were anesthetized with a cocktail of alpha chloralose (80 mg/kg) and urethane (800 mg/kg). Catheters were placed in the femoral artery and vein to record arterial and venous blood pressures. A PE 10 catheter was placed in the pericardial space for the injection of bradykinin (BK), an agent known to stimulate cardiac sympathetic afferents. In some rats, a balloon tipped catheter was placed in the right atrium to allow transient arrest of cardiac pumping for the estimation of mean circulatory filling pressure (MCFP), an estimate of systemic venous tone. In other rats, an electromagnetic flow probe was placed on the aortic arch for the direct measurement of cardiac output.In rats instrumented for the measurement of venous tone, pericardial injections of BK (0.4–20 μg/kg) were associated with dose‐related increases in mean arterial pressure (9±6 to 27±6 mm Hg) and MCFP (0.6±0.2 to 1.6±0.4 mm Hg). These responses were largely attenuated after ganglionic blockade. In rats instrumented for the measurement of cardiac output, pericardial injection of BK (10 μg/kg) increased mean arterial pressure (23±3 mm Hg) and cardiac output (16±2 ml/min).We interpret these data to indicate that activation of bradykinin‐sensitive pericardial afferents increases venous tone sufficiently to increase cardiac filling and cardiac output.Support or Funding InformationSupported by NIH R01 HL136741‐01 and the Basic Biomedical Sciences program of the Sanford School of Medicine, University of South Dakota.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.