Inhibitor Indomethacin Research Articles

COX Inhibition Increases Alternaria-Induced Pulmonary Group 2 Innate Lymphoid Cell Responses and IL-33 Release in Mice.

The cyclooxygenase (COX) metabolic pathway regulates immune responses and inflammation. The effect of the COX pathway on innate pulmonary inflammation induced by protease-containing fungal allergens, such as Alternaria alternata, is not fully defined. In this study, we tested the hypothesis that COX inhibition augments Alternaria-induced pulmonary group 2 innate lymphoid cell (ILC2) responses and IL-33 release. Mice were treated with the COX inhibitors indomethacin, flurbiprofen, or vehicle and challenged intranasally with Alternaria extract for four consecutive days to induce innate lung inflammation. We found that indomethacin and flurbiprofen significantly increased the numbers of ILC2 and IL-5 and IL-13 expression by ILC2 in the lung. Indomethacin also increased ILC2 proliferation, the percentages of eosinophils, and mucus production in the lung. Both indomethacin and flurbiprofen augmented the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation and that a COX product(s) inhibited IL-33 release. This is supported by the in vitro finding that the COX product PGE2 and the PGI2 analogs cicaprost decreased Alternaria extract-induced IL-33 release by human bronchial epithelial cells. Although contrasting effects of PGD2, PGE2, and PGI2 on ILC2 responses have been previously reported, the overall effect of the COX pathway on ILC2 function is inhibitory in Alternaria-induced innate airway inflammation.

Cellular mechanism underlying oxytocin-stimulated Cl- secretion in rat cauda epididymal epithelium.

The neurohypophyseal hormone oxytocin (OT) plays critical roles in lactation and parturition, while its function in male reproduction system is largely unknown. This study aims to investigate the effect of OT on regulating transepithelial ion transport in rat cauda epididymal epithelium. With the use of RT-PCR, Western blot, and immunohistochemical analysis, we found that OT receptor (OTR) was expressed and localized at the basal membrane of rat cauda epididymal epithelium. The short-circuit current (Isc) measurement showed that basolateral application of OT to the primary cultured rat cauda epididymal epithelial cells elicited an increase in Isc, which was abrogated by pretreating the epithelial cells with CFTRinh-172, a blocker of cystic fibrosis transmembrane conductance regulator (CFTR). Pretreatment with the prostaglandin H synthase inhibitors indomethacin and piroxicam, or the nonselective antagonists of prostaglandin E2 (PGE2) receptor EP2 or EP4, AH-6809, and AH-23848, significantly attenuated OT-stimulated Isc response. Furthermore, the generation of PGE2 was measured using enzyme-linked immunosorbent assay, demonstrating that OT induced a substantial increase in PGE2 release from primary cultured rat cauda epididymal epithelial cells. In conclusion, activation of OTR by OT triggered PGE2 release, resulting in CFTR-dependent Cl- secretion through paracrine/autocrine pathways in rat cauda epididymal epithelium.

Cardiorespiratory Fitness And The Cerebrovascular Response To A Metabolic Stimulus Following Cyclooxygenase Inhibition

Cardiorespiratory fitness (CRF) is positively associated with cerebrovascular function and cognition. We have previously shown that prostaglandins play an important role in regulating the cerebral vasodilator response to hypercapnia, and that the magnitude of change in cerebral vasodilator responses during cyclooxygenase (COX) inhibition is associated with CRF in older adults. However, it is unknown if CRF also influences the cerebrovascular response to a metabolic stimulus in older adults. PURPOSE: To determine the effects of CRF on the cerebrovascular response to a metabolic stimulus before and during COX inhibition in older adults. METHODS: Thirty-five participants completed a maximal exercise test on a cycle ergometer. Participants were split into two groups, High CRF (10 men, 8 women, age = 62 ± 5y) or Low CRF (7 men, 10 women, age = 66 ± 7y), based on the median VO2max (ml/kg/min). All participants completed two levels of the Stroop Color Word Test. Beat-to-beat mean arterial pressure (MAP) and middle cerebral artery velocity (MCAv) were measured at baseline and in response to each level of the Stroop test before and after administration of the COX inhibitor Indomethacin (INDO). The maximum MAP, MCAv, and cerebral pulsatility index (PI) responses were calculated as the highest 3-beat average during each cognitive challenge. RESULTS: There were no differences between high and low CRF groups in MCAv at rest or in response to the metabolic stimulus. There was a trend for lower PI at rest (p = 0.09) and in response to the Stroop test (p = 0.09) in the high CRF group compared with the low CRF group. During INDO, MCAv decreased (Low CRF: -29 ± 4%, High CRF: -27 ± 3%; p < 0.01) and PI increased (Low CRF: 22 ± 3%, High CRF: 17 ± 3%; p < 0.01). During INDO, MCAv at rest was not different between groups; however, PI was lower in the high CRF compared to low CRF group (Low CRF: 0.98 ± 0.05, High CRF: 0.87 ± 0.03; p < 0.05). Lastly, the change in MCAv and PI in response to the metabolic stimulus did not differ between groups. CONCLUSION: In older adults, elevated levels of CRF may lead to a lower PI at rest and in response to a metabolic stimulus. Additionally, COX inhibition did not alter the cerebrovascular response to a metabolic stimulus. Supported by NIH Grant HL118154.

Tert-Butyl Hydroperoxide Stimulated Apoptosis Independent of Prostaglandin E2 and IL-6 in the HTR-8/SVneo Human Placental Cell Line.

Significant gaps exist in our knowledge of how cellular redox status, sometimes referred to as oxidative stress, impacts placental trophoblasts. The present study used tert-butyl hydroperoxide (TBHP) as a known generator of reactive oxygen species (ROS) in the extravillous trophoblast cell line HTR-8/SVneo to examine the role of cellular redox disruption of prostaglandin E2 (PGE2) and the cytokine IL-6 in cell death. Cells were exposed to 0, 12.5, 25, or 50μM TBHP for 4, 8, and 24h to ascertain effects on cell viability, caspase 3/7 activity, PGE2 release, PTGS2 mRNA expression, and IL-6 release. Experiments with inhibitors included the cyclooxygenase inhibitor indomethacin, mitogen-activated protein kinase inhibitors (PD169316, U0126, or SP600125), or treatments to counter expected consequences of TBHP-stimulated generation of ROS (deferoxamine [DFO], butylated hydroxyanisole [BHA], and N,N'-diphenyl-1,4-phenylenediamine [DPPD]) using 24-h exposure to 50μM TBHP. Cell viability, measured by ATP content, decreased 24% relative to controls with a 24-h exposure to 50μM TBHP, but not at lower TBHP concentrations nor at earlier time points. Exposure to 50μM TBHP increased caspase 3/7 activity, an indicator of apoptosis, after 8 and 24h. Antioxidant treatment markedly reduced TBHP-stimulated caspase 3/7 activity, PGE2 release, and IL-6 release. TBHP-stimulated IL-6 release was blocked by PD169316 but unaltered by indomethacin. These data suggest that TBHP-stimulated IL-6 release and caspase 3/7 activation were independent of PGE2 yet were interrupted by treatments with known antioxidant properties, providing new insight into relationships between PGE2, IL-6, and apoptosis under conditions of chemically induced cellular oxidation.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar–Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

Characterization of the intestinal absorption of morroniside from Cornus officinalis Sieb. et Zucc via a Caco-2 cell monolayer model.

Morroniside is a biologically active polyphenol found in Cornus officinalis Sieb. et Zucc (CO) that exhibits a broad spectrum of pharmacological activities, such as protecting nerves, and preventing diabetic liver damage and renal damage. However, little data are available regarding the mechanism of its intestinal absorption. Here, an in vitro human intestinal epithelial cell model of cultured Caco-2 cells was applied to study the absorption and transport of morroniside. The effects of donor concentration, pH and inhibitors were investigated. The bidirectional permeability of morroniside from the apical (AP) to the basolateral (BL) side and in the reverse direction was studied. When administered at three tested concentrations (5, 25 and 100 μM), the apparent permeability coefficient (Papp) values in the AP-to-BL direction ranged from 1.59 × 10−6 to 2.66 × 10−6 cm/s. In the reverse direction, BL-to-AP, the value was ranged from 2.67 × 10−6 to 4.10 × 10−6 cm/s. The data indicated that morroniside transport was pH-dependent. The permeability of morroniside was affected by treatment with various inhibitors, such as multidrug resistance protein inhibitors MK571 and indomethacin, as well as the breast cancer resistance protein inhibitor apigenin. The mechanisms of the intestinal absorption of morroniside may involve multiple transport pathways, such as the passive diffusion and efflux protein-mediated active transport especially involving multidrug resistance protein 2 and breast cancer resistance protein. After the addition of CO, the Papp values in the AP-to-BL direction increased significantly, therefore, it can be assumed that some ingredients in the CO promote morroniside absorption in the small intestine.

Antiplatelet properties of Pim kinase inhibition are mediated through disruption of thromboxane A2 receptor signaling.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumor development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 kinase is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired hemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function were non-additive to inhibition caused by the cyclo-oxygenase inhibitor indomethacin or the thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase-dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of cyclo-oxygenase-1 inhibition or direct antagonism of the thromboxane A2 receptor that, while attenuating thrombosis, does not increase bleeding.

Role of Arginase in Selective Impairment of Endothelium-Dependent Nitric Oxide Synthase-Mediated Dilation of Retinal Arterioles during Early Diabetes.

PurposeRetinal vasomotor activity can be regulated by two major endothelial enzymes, nitric oxide synthase (NOS) and cyclooxygenase (COX). The vascular arginase also consumes a NOS substrate and thus impedes NOS-mediated vasodilation. Diabetes mellitus exhibits vascular complications in the retina with elevated oxidative stress and compromised NOS-mediated vasodilation. However, the underlying molecular mechanisms remain unclear, and the effect of diabetes on COX-mediated vasodilation is unknown. Herein, we examined the relative impact of diabetes on retinal arteriolar dilations to COX and NOS activation and the roles of arginase and superoxide in diabetes-induced vasomotor dysfunction.MethodsRetinal arterioles were isolated from streptozocin-induced diabetic pigs (2 weeks of hyperglycemia, 433 ± 27 mg/dL) or age-matched control pigs (97 ± 4 mg/dL). The vasodilations to bradykinin (NOS activator) and histamine (NOS/COX activator) were examined in vitro.ResultsRetinal arteriolar dilations to histamine and bradykinin were significantly reduced after 2 weeks of diabetes. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated the dilations of control vessels, but not diabetic vessels, to histamine. In the presence of L-NAME and COX inhibitor indomethacin, histamine-induced dilations of control and diabetic vessels were reduced similarly. Treatment of diabetic vessels with arginase inhibitor nor-NOHA, but not superoxide dismutase mimetic TEMPOL, preserved both histamine- and bradykinin-induced dilations in an L-NAME-sensitive manner.ConclusionsArginase, rather than superoxide, impairs endothelium-dependent NOS-mediated dilation of retinal arterioles during diabetes, whereas vasodilation mediated by COX remains intact. Blockade of vascular arginase may improve endothelial function of retinal arterioles during early onset of diabetes.

Nucleoside transporters are critical to the uptake and antioxidant activity of 7,8-dihydroneopterin in monocytic cells

7,8-Dihydroneopterin protects cells intracellularly from oxidative stress-induced death, but its mode of transport across the cell membrane is unknown. Nucleosides, such as guanosine, are transported via nucleoside transporters of the equilibrative and concentrative forms. Therefore, the objective of this study was to identify which membrane transporters are responsible for 7,8-dihydroneopterin transport in cells and whether this is necessary for protection against oxidative stress. Monocytic cell lines U937, THP-1 and human monocytes were incubated with varying concentrations of 7,8-dihydroneopterin with or without nucleoside transporter inhibitors nitrobenzylthioinosine (NBMPR; ENT1), dipyridamole (DP; ENT1 and ENT2) or indomethacin (INDO; CNT). Only DP inhibited 7,8-dihydroneopterin uptake in U937 cells, while NBMPR and DP inhibited 7,8-dihydroneopterin uptake in THP-1 cells. All three inhibitors limited 7,8-dihydroneopterin uptake in human monocytes at short time points only. When the cells were incubated with 10 mM of the peroxyl radical generator 2,2'-azobis-2-methyl-propanimidamide, dihydrochloride (AAPH) a 50–80% loss of cell viability was measured. 7,8-dihydroneopterin protected all cell lines against AAPH-induced cell death, which was prevented with DP in U937 cells, NBMPR in THP-1 cells and a combination of all three nucleoside inhibitors in human monocytes. These data indicate 7,8-dihydroneopterin is transported across the cell membrane of monocytic cells via equilibrative and concentrative nucleoside transporters in a cell lineage-dependent manner. The data also indicate protection from peroxyl radical-generated cell death with 7,8-dihydroneopterin is intracellular and facilitated through nucleoside transporters in monocytic cells.

COX inhibition enhances allergen-induced IL-33 release and ILC2 responses in mouse lungs

Abstract The cyclooxygenase (COX) metabolic pathway has regulatory functions in immune responses and inflammation. However, the effect of the COX pathway on innate airway inflammation induced by protease-containing allergens such as Alternaria alternata is not fully defined. Here we show that COX inhibition augmented innate lung type 2 responses after repeated airway exposures to Alternaria extract in mice. We treated wild type BALB/c and IL-33 KO mice with either the COX inhibitor indomethacin or vehicle in drinking water and challenged mice intranasally with Alternaria extract for 4 consecutive days to induce innate lung inflammation. We found that indomethacin significantly increased the numbers of group 2 innate lymphoid cells (ILC2) and IL-5+IL-13+ ILC2 in the lung. Indomethacin also increased type 2 cytokine (IL-5 and IL-13) responses, the percentages of eosinophils, and mucus production in the lung. IL-33 is required for Alt-induced lung ILC2 responses, and indomethacin did not change IL-5 and IL-13 expression in IL-33 KO mice. Consistently, indomethacin increased the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation. Indomethacin also increased reactive oxygen species (ROS) production in the lung, providing a possible mechanism for the increased IL-33 release. Although contrasting effects of PGD2 and PGE2/PGI2 on ILC2 responses have been previously reported, the overall effect of COX pathway on ILC2 function is inhibitory in Alternaria extract-induced airway innate type 2 responses.

Comparison of the effect of two drugs paracetamol with intravenous ibuprofen in patent ductus arteriosus treatment

Background: Patent ductus arteriosus (PDA) is a common cause of morbidity and mortality among premature infants that affects more than 40% of them. PDA treatment includes medical and surgical treatment. Most drugs used to block PDA are cyclooxygenase inhibitors (ibuprofen and indomethacin). The role of paracetamol as an alternative therapy in PDA ligation has been considered in recent years due to the potential side effects of cyclooxygenase inhibitors.Methods: Patients in the first group were treated with intravenous paracetamol at a dose of 15 mg/kg every 6 hours for three days and the second group was treated with intravenous ibuprofen at a daily dose of 10 mg/kg daily and 5 mg/kg for the second and third days. At the end of the treatment period (day 3), they underwent echocardiography again. If the echocardiographic findings indicated no closure of the arterial duct, patients were treated with the aforementioned drug for another period and rechecked at the end of the third day, and at each stage required information was collected.Results: Arterial duct closure in paracetamol group was 96.7% and in ibuprofen group was 100%. The effects of both paracetamol and ibuprofen were similar in terms of renal parameters but in terms of effects on liver parameters. The effect of paracetamol on all liver parameters except aspartate transaminase (AST) was significant, but ibuprofen was able to affect only bilirubin among liver parameters and had no significant effect on both AST and alanine aminotransferase, parameters.Conclusions: Results showed that both paracetamol and ibuprofen are effective in treating of PDA and had similar impact.

Puerarin Inhibits Endothelium-Dependent Contractions in Mouse Carotid Arteries.

BackgroundMany bioactive ingredients of medicinal plants are known to produce vaso-protective benefits. Puerarin is one of the major isoflavone glucosides found in the root of kudzu vine and it exerts an anti-inflammatory effect and many other pharmacological actions. However, the mechanism underlying the vascular effect of puerarin is incompletely understood. Therefore, the present study aims to examine how puerarin reduces endothelium-dependent contractions (EDCs) in mouse arteries.Material/MethodsEDCs were evoked by acetylcholine (ACh) in isolated mouse carotid arteries with intact endothelium pretreated with Nω-NO2-L-Arg-OMe (L-NAME). The arteries were pretreated with puerarin and other pharmacological inhibitors before the addition of cumulative concentrations of ACh. The concentration of several prostaglandins (PGs) was measured by high performance liquid chromatography-coupled spectrometry (HPLC-MS).ResultsEDCs induced by ACh only presented in endothelium-intact arteries pretreated by L-NAME and EDCs were prevented by the treatment with cyclooxygenase (COX) inhibitor indomethacin (3 μmol/L) or thromboxane prostanoid receptor (TP receptor) antagonist S18886 (30 nmol/L). Acute 40-minute treatment with puerarin reduced EDCs in a concentration-dependent manner without affecting U46619-induced contraction. However, treatment with puerarin did not inhibit ACh-induced production of prostaglandins (PGs) in endothelium-intact arteries.ConclusionsThe present results show that puerarin is able to suppress EDCs in mouse carotid arteries, independent of inhibition of TP receptor or COX2-derived PGs.

NADPH Oxidase 2 and 4 Contribute to Endothelium‐Dependent Dilation in Healthy Human Arterioles

NADPH oxidases (NOXs) are a major source of reactive oxygen species (ROS) in the vasculature and play diverse roles in vascular health and disease. We have recently shown that NOX‐derived ROS are involved in flow‐induced dilation of human arterioles from subjects with coronary artery disease (CAD). However, the role of NOXs in non‐diseased human arterioles remains largely unclear. Previous studies indicate that NOXs contribute to endothelium‐dependent vasodilation in normal animal arteries. This study is designed to test whether NOX2 and NOX4 play a role in receptor and non‐receptor mediated endothelium‐dependent vasodilation in human adipose arterioles (HAA). HAAs (~150 μm ID), isolated from fat tissues from patients without coronary artery disease (non‐CAD) or &lt;1 risk factor, were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. In arterioles constricted with endothelin‐1, acetylcholine (Ach; 10−9 to 10−5 M) elicited concentration‐dependent dilation (maximal dilation at 10−5 M: 95±3%, n=4), and this dilation was reduced in the presence of nitric oxide (NO) synthase inhibitor nitro‐L‐arginine methyl ester (L‐NAME) and cyclooxygenase inhibitor indomethacin (Indo) (maximal dilation: 63±7%, n=4). Ach‐induced dilation was inhibited by NOX2 inhibitor GSK2795039 (10−6 M; maximal dilation: 57±10%, n=5) and to a less extent by NOX4 inhibitor GKT137831 (10−6 M; maximal dilation: 86±4%, n=5). In the presence of L‐NAME and Indo, NOX2 and NOX4 inhibitors largely abolished Ach‐induced vasodilation (maximal dilation: 29±9% and 18±5%, respectively, n=4). We also examined the effect of NOX inhibition on non‐receptor‐mediated concentration‐dependent dilation by the Ca2+ ionophore A23187 (10−11 to 10−7 M). A23187‐induced dilation (maximal dilation at 10−7 M: 68±5%, n=4) was inhibited by NOX2 (maximal dilation: 6±2%, n=4) and NOX4 (maximal dilation: 15±2%, n=5). Unexpectedly, L‐NAME and Indo also inhibited A23187‐dependent dilation (maximal dilation: 7±2%, n=4). Together, these data support our hypothesis that NOX2 and NOX4 are involved in receptor and non‐receptor mediated endothelium‐dependent vasodilation in non‐CAD HAA. While A23187 dilation is largely NO/prostacyclin (PGI2)‐dependent, there is a non‐NO/PGI2 component contributing to NOX inhibitor‐sensitive vasodilator response to Ach.Support or Funding InformationSupport for this research was provided by the National Heart, Lung and Blood Institute Grant R01‐HL 096647. Tissue was supplied by Cardiovascular Surgery Associates, Midwest Heart Surgery Institute, Wisconsin Heart Group, and Froedtert Memorial Lutheran Hospital.

Sex Differences in the Cerebrovascular Response to a Metabolic Stimulus

BackgroundBlood pressure control and regulation of cerebral blood flow are influenced by biological sex. We have previously demonstrated sex differences in cerebrovascular reactivity to hypercapnia in young adults. However, sex differences in the cerebrovascular response to a metabolic stimulus are unknown. As prostaglandins play a role in regulating cerebrovascular reactivity, evaluating cerebrovascular responses to a metabolic stimulus with cyclooxygenase (COX) inhibition may reveal important differences between males and females. Therefore, the purpose of this study was to investigate sex differences in the cerebrovascular response to a metabolic stimulus with and without COX inhibition in young adults.MethodsForty‐three young adults, 21 males (age = 28 ± 5 y) and 22 females (age = 25 ± 5 y), completed two levels of difficulty of the Stroop Color Word Test and the average response was calculated. Beat‐to‐beat mean arterial pressure (MAP) and middle cerebral artery velocity (MCAv) were measured at baseline and in response to each level of the Stroop test before and after administration of the COX inhibitor indomethacin. The peak MAP, MCAv, pulse pressure (PP), and cerebral pulsatility index (PI) responses were calculated as the highest 3‐beat average during each Stroop test.ResultsAt rest, MCAv was higher (Females: 65 ± 2, Males: 55 ± 2 cm/s, p &lt; 0.01) and PI was lower (Females: 0.70 ± 0.02, Males: 0.78 ± 0.02, p &lt; 0.05) in females compared to males, with no differences in MAP or PP. There were no differences in the percent change or peak MCAv, MAP, PP, or PI between males and females in response to the metabolic stimulus. COX inhibition reduced MCAv (Females: −28 ± 2%, Males: −28 ± 3%, p &lt; 0.01 compared to baseline) and increased PI (Females: 17 ± 2%, Males: 20 ± 4%, p &lt; 0.01 compared to baseline), with no change in MAP or PP. During COX inhibition, MCAv at rest was higher (Females: 46 ± 2, Males: 38 ± 1 cm/s, p &lt; 0.01) and PI was lower (Females: 0.81 ± 0.03, Males: 0.93 ± 0.03, p &lt; 0.05) in females compared to males, with no differences in MAP or PP. During COX inhibition, the percent change in MCAv, MAP, and PI in response to the metabolic stimulus did not differ between groups (p &gt; 0.05); however, the percent change in PP during the metabolic stimulus was greater in males compared to females (Males: 16 ± 2%, Females: 9 ± 1%, p &lt; 0.01).ConclusionYoung females had greater MCAv and lower PI at rest and this difference persisted following COX inhibition. Prior to COX inhibition, there were no differences between males and females in response to the metabolic stimulus. After COX inhibition, young males demonstrated a greater change in PP in response to the metabolic stimulus. These results suggest that COX inhibition reveals sex differences in the PP response to a metabolic stimulus.Support or Funding InformationFundingThis research was funded by NIH grant HL118154.

Cyclooxygenase Inhibition Increases the Sympathetic Response to Hypercapnia

Cyclooxygenase (COX) catalyzes the formation of prostaglandins and influences blood flow regulation. Previous studies have shown that COX inhibition reduces cerebral blood flow velocity and cerebrovascular reactivity to CO2 (CVR), however, the effects of COX inhibition on neurovascular regulation is unknown. Sympathetic nerve activity is an important modulator of blood pressure and blood flow. The purpose of this study was to determine the effect of COX inhibition on the muscle sympathetic nerve activity (MSNA) response to hypercapnia during CVR testing.MethodsFifteen healthy adults were evaluated (M/F = 7/8; age = 40 ± 21 years; BMI = 24 ± 2 kg/m2) before (Pre) and 90 minutes after oral administration of the COX inhibitor indomethacin (Indo), given at 1.2mg/kg. Mean arterial pressure (MAP), heart rate (HR) and MSNA were measured continuously throughout the protocol. After baseline measurements were recorded, participants were evaluated breathing 6% CO2. Results are reported as mean ± SE.ResultsAt baseline, HR was 54 ± 2 bpm, MAP was 100 ± 4 mmHg, MSNA burst frequency was 24 ± 2 bursts/min and MSNA burst incidence was 45 ± 4 bursts/100hb. The change in HR when exposed to hypercapnia was not different after Indo (Pre: 6 ± 2 bpm vs. Indo: 4 ± 1 bpm; p&gt;0.05). The change in MAP (Pre: 0 ± 2 mmHg vs. Indo: 6 ± 1 mmHg; p&lt;0.05), MSNA burst frequency (Pre: 1 ± 1 bursts/min vs. Indo: 4 ± 1 bursts/min; p&lt;0.05), and MSNA burst incidence (Pre: −3 ± 2 bursts/100hb vs. Indo: 4 ± 2 bursts/100hb; p&lt;0.05) was greater after Indo administration.ConclusionThe sympathetic response to hypercapnia was greater following the administration of Indo. Taken together, these results suggest that hypercapnia‐induced sympathoexcitation may be altered following COX inhibition. Future studies could evaluate if the sympathetic responses to hypercapnia vary with age or sex.Support or Funding InformationNIH HL118154

Transient receptor potential ankyrin-1 causes rapid bronchodilation via nonepithelial PGE2.

Transient receptor potential ankyrin-1 (TRPA1) is a ligand-gated cation channel that responds to endogenous and exogenous irritants. TRPA1 is expressed on multiple cell types throughout the lungs, but previous studies have primarily focused on TRPA1 stimulation of airway sensory nerves. We sought to understand the integrated physiological airway response to TRPA1 stimulation. The TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) were tested in sedated, mechanically ventilated guinea pigs in vivo. Reproducible bronchoconstrictions were induced by electrical stimulation of the vagus nerves. Animals were then treated with intravenous AITC or CINN. AITC and CINN were also tested on isolated guinea pig and mouse tracheas and postmortem human trachealis muscle strips in an organ bath. Tissues were contracted with methacholine, histamine, or potassium chloride and then treated with AITC or CINN. Some airways were pretreated with TRPA1 antagonists, the cyclooxygenase inhibitor indomethacin, the EP2 receptor antagonist PF 04418948, or tetrodotoxin. AITC and CINN blocked vagally mediated bronchoconstriction in guinea pigs. Pretreatment with indomethacin completely abolished the airway response to TRPA1 agonists. Similarly, AITC and CINN dose-dependently relaxed precontracted guinea pig, mouse, and human airways in the organ bath. AITC- and CINN-induced airway relaxation required TRPA1, prostaglandins, and PGE2 receptor activation. TRPA1-induced airway relaxation did not require epithelium or tetrodotoxin-sensitive nerves. Finally, AITC blocked airway hyperreactivity in two animal models of allergic asthma. These data demonstrate that stimulation of TRPA1 causes bronchodilation of intact airways and suggest that the TRPA1 pathway is a potential pharmacological target for bronchodilation.

P2X3 and P2X2/3 receptors activation induces articular hyperalgesia by an indirect sensitization of the primary afferent nociceptor in the rats’ knee joint

We have previously shown that endogenous adenosine 5′-triphosphate (ATP), via P2X3 and P2X2/3 receptors, plays an essential role in carrageenan-induced articular hyperalgesia model in rats' knee joint. In the present study, we used the rat knee joint incapacitation test, Enzyme-Linked Immunosorbent Assay (ELISA), and myeloperoxidase enzyme activity assay, to test the hypothesis that the activation of P2X3 and P2X2/3 receptors by their agonist induces articular hyperalgesia mediated by the inflammatory mediators bradykinin, prostaglandin, sympathomimetic amines, pro-inflammatory cytokines and by neutrophil migration. We also tested the hypothesis that the activation of P2X3 and P2X2/3 receptors contributes to the articular hyperalgesia induced by the inflammatory mediators belonging to carrageenan inflammatory cascade. The non-selective P2X3 and P2X2/3 receptors agonist αβ-meATP induced a dose-dependent articular hyperalgesia, which was significantly reduced by the selective antagonists for P2X3 and P2X2/3 receptors (A-317491), bradykinin B1- (DALBK) or B2-receptors (bradyzide), β1-(atenolol) or β2-adrenoceptors (ICI-118,551), by the pre-treatment with cyclooxygenase inhibitor (indomethacin) or with the nonspecific selectin inhibitor (Fucoidan). αβ-meATP induced the release of pro-inflammatory cytokines TNFα, IL-1β, IL-6, and CINC-1, as well as the neutrophil migration. Moreover, the co-administration of A-317491 significantly reduced the articular hyperalgesia induced by bradykinin, prostaglandin E2 (PGE2), and dopamine. These findings suggest that peripheral P2X3 and P2X2/3 receptors activation induces articular hyperalgesia by an indirect sensitization of the primary afferent nociceptor of rats' knee joint through the release of inflammatory mediators. Further, they also indicate that the activation of these purinergic receptors by endogenous ATP mediates the bradykinin-, PGE2-, and dopamine-induced articular hyperalgesia.

Shenfu Injection Promotes Vasodilation by Enhancing eNOS Activity Through the PI3K/Akt Signaling Pathway In Vitro

Vasomotor dysfunction is one of the key pathological aspects of shock and heart failure (HF). Shenfu injection (SFI) has been widely used for the treatment of shock and HF in China. Pharmacological studies have suggested that SFI can reduce peripheral circulation resistance and improve microcirculation. However, whether it has a regulatory effect on macrovascular has not been elucidated. In this study, we used thoracic aorta rings isolated from Wistar rats and the human umbilical vein cell line (EA.hy926) to explore the vasodilative activity of SFI and its potential mechanisms. The relaxation due to SFI was measured after pre-treatment with selective soluble guanylate cyclase (sGC) inhibitor or cyclooxygenase (COX) inhibitor and compared with the vasodilation effect of SFI only treated with norepinephrine (NE). The contents of NO, endothelin-1 (ET-1), endothelial nitric oxide synthase (eNOS), COX-1, 6-K-PGF1α, and caveolin-1 were evaluated respectively. Additionally, the level of eNOS mRNA and total eNOS and its phosphorylation were studied to investigate the potential mechanisms involved. Experimental results showed that SFI markedly attenuated NE-induced vasoconstriction but that this effect was significantly eliminated after pre-incubation with the selective sGC inhibitor 1-H-[1, 2, 4] oxadiazolo [4, 3-α] quinoxaline-1-one (ODQ), instead of the COX inhibitor indomethacin (INDO). SFI significantly increased the eNOS content and up-regulated the eNOS mRNA expression, while it did not affect the content of COX-1 and 6-K-PGF1α. SFI also markedly increased NO content but significantly reduced the content of ET-1 and caveolin-1 in the cell supernatant. Furthermore, it promoted the expression of total eNOS and the phosphorylation of eNOS at serine (Ser) 1177 but inhibited the phosphorylation at threonine (Thr) 495, which was significantly reversed by PI3K-specific inhibitor LY294002. In conclusion, our study showed the vasodilation effect of SFI in thoracic aorta is mediated entirely by enhancing eNOS activity through the PI3K/Akt signaling pathway, providing novel knowledge on the effect of SFI on shock and HF for future clinical applications.

Upregulation of Cytokines and Differentiation of Th17 and Treg by Dendritic Cells: Central Role of Prostaglandin E2 Induced by Mycobacterium bovis

Mycobacterium bovis (M. bovis) is a zoonotic pathogen that causes bovine and human tuberculosis. Dendritic cells play a critical role in initiating and regulating immune responses by promoting antigen-specific T-cell activation. Prostaglandin E2 (PGE2)-COX signaling is an important mediator of inflammation and immunity and might be involved in the pathogenesis of M. bovis infection. Therefore, this study aimed to reveal the character of PGE2 in the differentiation of naïve CD4+ T cells induced by infected dendritic cells (DCs). Murine bone marrow-derived DCs were pre-infected with M. bovis and its attenuated strain M. bovis bacillus Calmette-Guérin (BCG). Then, the infected DCs were co-cultured with naïve CD4+ T cells with or without the cyclooxygenase (COX) inhibitor indomethacin. Quantitative RT-PCR analysis and protein detection showed that PGE2/COX-2 signaling was activated, shown by the upregulation of PGE2 production as well as COX-2 and microsomal PGE2 synthase (mPGES1) transcription in DCs specifically induced by M. bovis and BCG infection. The further co-culture of infected DCs with naïve CD4+ T cells enhanced the generation of inflammatory cytokines IL-17 and IL-23, while indomethacin suppressed their production. Following this, the differentiation of regulatory T cells (Treg) and Th17 cell subsets was significantly induced by the infected DCs rather than uninfected DCs. Meanwhile, M. bovis infection stimulated significantly higher levels of IL-17 and IL-23 and the differentiation of Treg and Th17 cell subsets, while BCG infection led to higher levels of TNF-α and IL-12, but lower proportions of Treg and Th17 cells. In mice, M. bovis infection generated more bacterial load and severe abnormalities in spleens and lungs, as well as higher levels of COX-2, mPGE2 expression, Treg and Th17 cell subsets than BCG infection. In conclusion, PGE2/COX-2 signaling was activated in DCs by M. bovis infection and regulated differentiation of Treg and Th17 cell subsets through the crosstalk between DCs and naive T cells under the cytokine atmosphere of IL-17 and IL-23, which might contribute to M. bovis pathogenesis in mice.

Oral Intake of EPA:DHA 6:1 by Middle-Aged Rats for One Week Improves Age-Related Endothelial Dysfunction in Both the Femoral Artery and Vein: Role of Cyclooxygenases.

In humans, aging is associated with endothelial dysfunction and an increased risk of venous thromboembolism. Although intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at a ratio of 6:1 by old rats improved the endothelial dysfunction in arteries, the impact on veins remains unclear. Eight-month-old male Wistar rats were either untreated or orally administered corn oil, EPA:DHA 1:1, or EPA:DHA 6:1 (500 mg/kg/d) for seven days. Vascular reactivity was studied by myography. In middle-aged femoral artery rings, acetylcholine caused a partial relaxation at low concentrations and a contractile response at high concentrations, whereas in the old femoral vein only a partial relaxation was observed. The EPA:DHA 6:1 treatment blunted the contractile response to acetylcholine in the middle-aged femoral artery and both EPA:DHA 6:1 and 1:1 increased the relaxation to acetylcholine in the old femoral vein. No such effects were observed with corn oil. Both the non-selective cyclooxygenase inhibitor indomethacin and the COX-1 inhibitor SC-560 increased the relaxation to acetylcholine in the middle-aged femoral artery whereas the COX-2 inhibitor NS-398 increased that in the middle-aged femoral vein. In conclusion, our results indicate that aging is associated with an endothelial dysfunction in the femoral artery and vein, which can be improved by EPA:DHA 6:1 treatment—most likely via a cyclooxygenase-dependent mechanism.