TNF/iNOS/NO pathway mediates host susceptibility to endothelial-dependent circulatory failure and death induced by betacoronavirus infection.

Poor disease outcomes and lethality are directly related to endothelial dysfunction in betacoronavirus infections. Here, we investigated the mechanisms underlying the vascular dysfunction caused by the betacoronaviruses MHV-3 and SARS-CoV-2. Wild-type C57BL/6 (WT) and knockout mice for inducible nitric oxide synthase (iNOS-/-) or TNF receptor 1 (TNFR1-/-) were infected with MHV-3, and K18-hACE2 transgenic mice expressing human ACE2 were infected with SARS-CoV-2. Isometric tension was used to evaluate vascular function. Protein expression was determined by immunofluorescence. Tail-cuff plethysmography and Doppler were used to assess blood pressure and flow, respectively. Nitric oxide (NO) was quantified with the DAF probe. ELISA was used to assess cytokine production. Survival curves were estimated using Kaplan-Meier. MHV-3 infection reduced aortic and vena cava contractility, arterial blood pressure, and blood flow, resulting in death. Resistance mesenteric arteries showed increased contractility. The contractility of the aorta was normalized by removing the endothelium, inhibiting iNOS, genetically deleting iNOS, or scavenging NO. In the aorta, iNOS and phospho-NF-kB p65 subunit expression was enhanced, along with basal NO production. TNF production was increased in plasma and vascular tissue. Genetic deletion of TNFR1 prevented vascular changes triggered by MHV-3, and death. Basal NO production and iNOS expression were also increased by SARS-CoV-2. In conclusion, betacoronavirus induces an endothelium-dependent decrease in contractility in macro-arteries and veins, leading to circulatory failure and death via TNF/iNOS/NO. These data highlight the key role of the vascular endothelium and TNF in the pathogenesis and lethality of coronaviruses.

Decreased expression of neuronal nitric oxide synthase contributes to the endothelial dysfunction associated with cigarette smoking in human.

Endothelial nitric oxide synthase (eNOS) malfunctioning has been proposed to contribute to the endothelial damage produced by cigarette. Besides eNOS, neuronal NOS (nNOS) is also expressed in most vascular tissues and plays an important role in the endothelium-dependent vascular relaxation. We hypothesize that nNOS may contribute to the endothelium dysfunction produced by cigarette in smokers. Vascular function was assessed in human resistance mesenteric arteries using a wire myograph, the level of protein expression by Western blot, eNOS and nNOS localization by immunofluorescence. Measurement of NO was assessed by fluorescence microscopy. Arteries of smokers showed impaired endothelium-dependent vascular relaxation in response to acetylcholine. Pharmacological nonselective blockade of NOS with l-NAME and selective nNOS blockade with inhibitor 1 reduced the relaxation of the mesenteric artery of both smokers and nonsmokers. Interestingly, the inhibitory effect of NOS inhibitors was greater in nonsmokers than in smokers. The expression of total nNOS and eNOS and the level of phosphorylation at eNOS-pSer1177 were reduced in arteries of smokers as compared with nonsmokers. No differences between groups were observed in the expression of total COX-1, COX-2, catalase and SOD-1. Immunofluorescence analysis showed the presence of nNOS in the vascular endothelium in both groups. Acetylcholine-induced NO production was impaired in arteries from smokers as compared to nonsmokers. Selective inhibition of nNOS caused a decreased in NO production, which was greater in nonsmokers than in smokers. Our data show that a decrease in nNOS expression contributes to the endothelial dysfunction caused by cigarette smoking in human.

The Cyclitol L-(+)-Bornesitol as an Active Marker for the Cardiovascular Activity of the Brazilian Medicinal Plant Hancornia speciosa.

The cyclitol bornesitol is the main constituent of the leaves from the antihypertensive medicinal plant Hancornia speciosa. This study aimed to investigate the ability of bornesitol to reduce blood pressure and its mechanism of action. Normotensive Wistar rats were divided into control group and bornesitol groups treated intravenously with bornesitol (0.1, 1.0 and 3.0 mg/kg). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded in non-anesthetized awake animals. Nitric oxide (NO) and angiotensin-converting enzyme (ACE) were measured in plasma by using colorimetric methods. Vascular reactivity study was performed in rat aorta rings and the involvement of nitric oxide synthase (NOS), calcium-calmodulin complex and phosphatidylinositol-3-kinase (PI3K)/Akt pathway in the vasodilator effect was investigated. Administration of bornesitol significantly reduced the SBP, increased the plasmatic level of nitrite, and decreased ACE activity in normotensive rats. In the rat aorta, bornesitol induced endothelium-dependent vasodilatation, which was abolished by NOS blockade. While calcium-calmodulin complex inhibition decreased the vasodilator effect of bornesitol, the inhibition of PI3K/Akt pathway did not alter it. Bornesitol reduced the blood pressure by a mechanism involving an increased production or bioavailability of NO, inhibition of ACE, and by an endothelium- and NO-dependent vasodilator effect. The present results support the use of bornesitol as an active marker for the cardiovascular activity of Hancornia speciosa.

Neuronal nitric oxide synthase contributes to the normalization of blood pressure in medicated hypertensive patients.

Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and besides NO, generates H2O2. nNOS has been proposed to contribute to the control of blood pressure in healthy humans. The aim of this study was to verify the hypothesis that nNOS can contribute to the control of vascular relaxation and blood pressure in hypertensive patients undergoing drug treatment. The study was conducted in resistance mesenteric arteries from 63 individuals, as follows: 1) normotensive patients; 2) controlled hypertensive patients (patients on antihypertensive treatment with blood pressure normalized); 3) uncontrolled hypertensive patients (patients on antihypertensive treatment that remained hypertensive). Only mesenteric arteries from uncontrolled hypertensive patients showed impaired endothelium-dependent vasorelaxation in response to acetylcholine (ACh). Selective nNOS blockade with inhibitor 1 and catalase, which decomposes H2O2, decreased vasorelaxation in the three groups. However, the inhibitory effect was greater in controlled hypertensive patients. Decreased eNOS expression was detected in both uncontrolled and controlled hypertensive groups. Interestingly nNOS expression and ACh-stimulated H2O2 production were greater in controlled hypertensive patients, than in the other groups. ACh-stimulated NO production was lower in controlled hypertensive when compared to normotensive patients, while uncontrolled hypertensive patients showed the lowest levels. Catalase and nNOS blockade inhibited ACh-induced H2O2 production. In conclusion, nNOS-derived H2O2 contributes to the endothelium-dependent vascular relaxation in human resistance mesenteric arteries. The endothelial dysfunction observed in uncontrolled hypertensive patients involves decreased eNOS expression and NO production. The normalization of vascular relaxation and blood pressure in controlled hypertensive patients involves increased nNOS-derived H2O2 and NO production.

Neuronal nitric oxide synthase-derived hydrogen peroxide effect in grafts used in human coronary bypass surgery.

Recently, H2O2 has been identified as the endothelium-dependent hyperpolarizing factor (EDHF), which mediates flow-induced dilation in human coronary arteries. Neuronal nitric oxide synthase (nNOS) is expressed in the cardiovascular system and, besides NO, generates H2O2 The role of nNOS-derived H2O2 in human vessels is so far unknown. The present study was aimed at investigating the relevance of nNOS/H2O2 signaling in the human internal mammary artery (IMA) and saphenous vein (SV), the major conduits used in coronary artery bypass grafting. In the IMA, but not in the SV, ACh (acetylcholine)-induced vasodilatation was decreased by selective nNOS inhibition with TRIM or Inhibitor 1, and by catalase, which specifically decomposes H2O2 Superoxide dismutase (SOD), which generates H2O2 from superoxide, decreased the vasodilator effect of ACh on SV. In the IMA, SOD diminished phenylephrine-induced contraction in endothelium-containing, but not in endothelium-denuded vessels. Importantly, while exogenous H2O2 produced vasodilatation in IMA, it constricted SV. ACh increased H2O2 production in both sets of vessels. In the IMA, the increase in H2O2 was inhibited by catalase and nNOS blockade. In SV, H2O2 production was abolished by catalase and reduced by nNOS inhibition. Immunofluorescence experiments showed the presence of nNOS in the vascular endothelium and smooth muscle cells of both the IMA and SV. Together, our results clearly show that H2O2 induced endothelium-dependent vascular relaxation in the IMA, whereas, in the SV, H2O2 was a vasoconstrictor. Thus, H2O2 produced in the coronary circulation may contribute to the susceptibility to accelerated atherosclerosis and progressive failure of the SV used as autogenous graft in coronary bypass surgery.

Endothelial dysfunction in DOCA-salt-hypertensive mice: role of neuronal nitric oxide synthase-derived hydrogen peroxide.

Endothelial dysfunction is a common problem associated with hypertension and is considered a precursor to the development of micro- and macro-vascular complications. The present study investigated the involvement of nNOS (neuronal nitric oxide synthase) and H2O2 (hydrogen peroxide) in the impaired endothelium-dependent vasodilation of the mesenteric arteries of DOCA (deoxycorticosterone acetate)-salt-hypertensive mice. Myograph studies were used to investigate the endothelium-dependent vasodilator effect of ACh (acetylcholine). The expression and phosphorylation of nNOS and eNOS (endothelial nitric oxide synthase) were studied by Western blot analysis. Immunofluorescence was used to examine the localization of nNOS and eNOS in the endothelial layer of the mesenteric artery. The vasodilator effect of ACh is strongly impaired in mesenteric arteries of DOCA-salt-hypertensive mice. Non-selective inhibition of NOS sharply reduced the effect of ACh in both DOCA-salt-hypertensive and sham mice. Selective inhibition of nNOS and catalase led to a higher reduction in the effect of ACh in sham than in DOCA-salt-hypertensive mice. Production of H2O2 induced by ACh was significantly reduced in vessels from DOCA-salt-hypertensive mice, and it was blunted after nNOS inhibition. The expression of both eNOS and nNOS was considerably lower in DOCA-salt-hypertensive mice, whereas phosphorylation of their inhibitory sites was increased. The presence of nNOS was confirmed in the endothelial layer of mesenteric arteries from both sham and DOCA-salt-hypertensive mice. These results demonstrate that endothelial dysfunction in the mesenteric arteries of DOCA-salt-hypertensive mice is associated with reduced expression and functioning of nNOS and impaired production of nNOS-derived H2O2 Such findings offer a new perspective for the understanding of endothelial dysfunction in hypertension.

Dihydrogoniothalamin, an Endothelium and NO-Dependent Vasodilator Drug Isolated from Aniba panurensis.

Dihydrogoniothalamin is a styrylpyrone isolated from the leaves of Aniba panurensis. The present work aimed at investigating the vasorelaxant activity of dihydrogoniothalamin and its underlying mechanism of action in the rat aorta. Dihydrogoniothalamin (0.01-100 µM) induced a concentration-dependent vasodilatation of aortas precontracted with phenylephrine. Endothelium removal or pretreatment of the preparation with NG nitro-L-arginine-methyl-ester abolished the vasodilator response for dihydrogoniothalamin. Pretreatment with calmidazolium did not affect the vasodilator response of dihydrogoniothalamin. On the other hand, wortmannin, a nonselective inhibitor of phosphatidylinositol 3-kinases, and protein kinase B inhibitor IV significantly shifted the concentration-response curve of dihydrogoniothalamin to the right and reduced its maximal effect. A nonselective antagonist of estrogen receptors, ICI 182,780, and a selective antagonist of estrogen receptor α, methyl-piperidino-pyrazole, were able to reduce the relaxation induced by dihydrogoniothalamin, but no effect was observed in the presence of the selective antagonists of estrogen receptor ß and G protein-coupled receptor 30, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP), and G-15, respectively. Dihydrogoniothalamin also increased the phosphorylation of the activation sites of endothelial nitric oxide synthase and protein kinase B. The present results led us to conclude that dihydrogoniothalamin is a vasodilator drug acting in an endothelium- and nitric oxide-dependent manner through a mechanism involving the activation of nitric oxide synthase via the phosphatidylinositol 3-kinase/protein kinase B pathway, partially by stimulation of estrogen receptor α.

Mechanism of the vasodilator effect of mono-oxygenated xanthones: a structure-activity relationship study.

The present study characterized the mechanisms involved in the vasodilator effect of two mono-oxygenated xanthones, 4-hydroxyxanthone and 4-methoxyxanthone. 9-Xanthenone, the base structure of xanthones, was used for comparison. 4-Hydroxyxanthone and 9-xanthenone induced a concentration-dependent and endothelium-independent vasodilator effect in arteries precontracted with phenylephrine (0.1 µmol ·â€ŠL-1) or KCl (50 mmol ·â€ŠL-1). 4-Methoxyxanthone induced a concentration-dependent vasodilator effect in arteries precontracted with phenylephrine, which was partially endothelium-dependent, and involved production of nitric oxide. In endothelium-denuded arteries precontracted with KCl, the vasodilator effect of 4-methoxyxanthone was abolished. The vasodilator effect of 4-hydroxyxanthone (96.22 ± 2.10 %) and 4-methoxyxanthone (96.57 ± 12.40 %) was significantly higher than observed with 9-xanthenone (53.63 ± 8.31 %). The presence of an oxygenated radical in position 4 made 4-hydroxyxanthone (pIC50 = 4.45 ± 0.07) and 4-methoxyxanthone (pIC50 = 5.04 ± 0.09) more potent as a vasodilator than 9-xanthenone (pIC50 = 3.92 ± 0.16). In addition, 4-methoxyxanthone was more potent than the other two xanthones. Ca2+ transients in vascular smooth muscle cells elicited by high K+ were abolished by 4-hydroxyxanthone and 9-xanthenone. The endothelium-independent effect of 4-methoxyxanthone was abolished by inhibition of K+ channels by tetraethylammonium. The current work shows that an oxygenated group in position 4 is essential to achieve Emax and to increase the potency of xanthones as vasodilators. Substitution of an OH by OCH3 in position 4 increases the potency of the vasodilator effect and changes the underling mechanism of action from the blockade of L-type calcium channels to an increase in NO production and activation of K+ channels.

Mechanism of the antihypertensive and vasorelaxant effects of the flavonoid tiliroside in resistance arteries.

Hypertension is a leading cause of death and disability globally, and its prevalence continues to accelerate. The cardiovascular effects of the flavonoid tiliroside have never been reported. In this work, using complementary in vivo and in vitro approaches, we describe the antihypertensive effect of tiliroside and the underlying mechanisms involved in the reduction of blood pressure. Tiliroside (1, 5 or 10 mg/kg) induced a dose-dependent long-lasting decrease in blood pressure in conscious DOCA-salt hypertensive rats that was accompanied by an increased heart rate. Tiliroside also induced a concentration-dependent vasodilation of mesenteric resistance arteries precontracted with phenylephrine. Removal of the endothelium or pretreatment of the preparation with L-NAME or indomethacin did not modify the vasodilator response for tiliroside. When vessels were precontracted with a high K⁺ (50 mM) solution, tiliroside exhibited a vasodilator effect similar to that observed in vessels precontracted with phenylephrine. Experiments carried out in nominally Ca²âº-free solution showed that tiliroside antagonized CaCl2-induced contractions. Moreover, tiliroside reduced the rise in intracellular Ca²âº concentration induced by membrane depolarization in vascular smooth muscle cells. Finally, tiliroside decreased the voltage-activated peak amplitude of the L-type Ca²âº channel current in freshly dissociated vascular smooth muscle cells from mesenteric arteries. Altogether, our results point to an antihypertensive effect of tiliroside due to a reduction in peripheral resistance through blockage of voltage-activated peak amplitude of the L-type Ca²âº channel in smooth muscle cells.

Oral sub-chronic treatment with Terminalia phaeocarpa Eichler (Combretaceae) reduces liver PTP1B activity in a murine model of diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: The endemic Brazilian medicinal plants of the genus Terminalia (Combretaceae), popularly known as capitão, comprising the similar species Terminalia phaeocarpa Eichler and Terminalia argentea, are traditionally and indistinguishably used in the country to treat diabetes. AIM OF THE STUDY: The present work investigated the effect of 28 days of treatment with the crude ethanolic extract (CEE) and its derived ethyl acetate fraction (EAF) from T. phaeocarpa leaves in a mice model of diabetes. MATERIALS AND METHODS: Streptozotocin-nicotinamide-fructose diabetic model was used to evaluate the antidiabetic activity of 28 days of treatment with the CEE and EAF from the leaves of T. phaeocarpa and metformin as a positive control. Serum levels of total cholesterol, triglycerides, uric acid, ALP, AST, and ALT were measured with specific commercial kits and glucose with a strip glucometer. The thiobarbituric acid method measured the liver MDA level, while a colorimetric assay measured the GSH level and PTP1B activity. A UPLC-DAD profile was obtained to identify the main polyphenolic compound in the EAF. RESULTS: Treatment with CEE and EAF reduced plasma glucose in diabetic mice. At the end of the treatment, the plasma glucose level was significantly lower in EAF-treated (100 mg/kg) diabetic mice (106.1 ± 13.7 mg/dL) than those treated with 100 mg/kg CEE (175.2 ± 20.9 mg/dL), both significantly lower than untreated diabetic mice (350.4 ± 28.1 mg/dL). The serum levels of total cholesterol, triglycerides, uric acid, ALP, AST, and ALT were significantly reduced in diabetic mice treated with CEE and EAF. In the livers of diabetic mice, the treatment with CEE and EAF reduced MDA levels and the activity of the enzyme PTP1B (96.9 ± 3.7%, 113.8 ± 2.8%, and 134.8 ± 4.6% for CEE-, EAF-treated, and untreated diabetic mice, respectively). Galloylpunicalagin was the main polyphenol observed in the EAF of T. phaeocarpa. CONCLUSION: The present results demonstrate the significant antidiabetic effect of CEE and EAF of T. phaeocarpa and their reduction on the markers of liver dysfunction in diabetic mice. Moreover, the antidiabetic activity of T. phaeocarpa might be associated with lowering the augmented activity of the PTP1B enzyme in the liver of diabetic mice.

Involvement of the L-arginine/nitric oxide/cyclic guanosine monophosphate pathway in peripheral antinociception induced by N-palmitoyl-ethanolamine in rats.

N-palmitoyl-ethanolamine (PEA) is an endogenous substance that was first identified in lipid tissue extracts. It has been classified as a CB(2) receptor agonist. Exogenous PEA has the potential to become a valid treatment for neuropathic and inflammatory pain. In spite of the well-demonstrated antiinflammatory properties of PEA, its involvement in controlling pain pathways remains poorly characterized. The participation of the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway in peripheral antinociception has been established by our group to the µ-, κ- or δ-opioid receptor agonists, nonsteroidal analgesics, α(2C) -adrenoceptor agonists, and even nonpharmacological electroacupuncture. The aim of this study was to verify whether the peripheral antinociception effects of PEA involve the activation of this pathway. All drugs were locally administered to the right hind paw of male Wistar rats. The paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E(2) . PEA elicited a local peripheral antinociceptive effect that was antagonized by the nonselective NO synthase (NOS) inhibitor L-NOARG and the selective neuronal NOS (nNOS) inhibitor L-NPA. Selective inhibition of endothelial (eNOS) and inducible (iNOS) NOS via L-NIO and L-NIL, respectively, was ineffective at blocking the effects of a local PEA injection. In addition, the dosage of nitrite in the homogenized paw, as determined by colorimetric assay, indicated that exogenous PEA is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ antagonized the PEA effect, whereas the cGMP-phosphodiesterase inhibitor zaprinast potentiated the antinociceptive effect of low-dose PEA. This study provides evidence that PEA activates nNOS, thus initiating the NO/cGMP pathway and inducing peripheral antinociceptive effects.

Inhibition of α-glucosidase and hypoglycemic effect of stilbenes from the Amazonian plant Deguelia rufescens var. urucu (Ducke) A. M. G. Azevedo (Leguminosae).

The control of blood glucose levels is critical in the treatment of diabetes mellitus. α-Glucosidase inhibitors are of great importance in reducing hyperglycemia, and plants have provided many of these agents. The present study aimed at investigating the effect of two stilbenes, lonchocarpene and 3,5-dimethoxy-4'-O-prenyl-trans-stilbene (DPS), isolated from the Amazonian plant Deguelia rufescens var. urucu, on α-glucosidase activity and on mice postprandial hyperglycemia. Lonchocarpene and DPS inhibited α-glucosidase in vitro, with pIC(50) values of 5.68 ± 0.12 and 5.73 ± 0.08, respectively. In addition, when given orally, DPS produced a significant reduction of hyperglycemia induced by an oral tolerance test, while lonchocarpene did not. Data suggest that DPS may have a potential use as an antidiabetic drug.

Definition of chemical markers for Hancornia speciosa Gomes by chemometric analysis based on the chemical composition of extracts, their vasorelaxant effect and α-glucosidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Hancornia speciosa Gomes (Apocynaceae) is a tree found in the Brazilian savannah, traditionally used to treat several diseases, including diabetes and hypertension. The anti-hypertensive activity of H. speciosa leaves (HSL) has been demonstrated in different models and is credited to the vasodilator effect and ACE (angiotensin-converting enzyme) inhibition. The hypoglycemic effect of HSL has been also reported. AIM OF THE STUDY: To establish correlations between the biological activities elicited by H. speciosa extracts and the contents of their major compounds, aiming to define chemical markers related to the potential antihypertensive and antidiabetic effects of the species. Additionally, it aimed to isolate and characterize the chemical structure of a marker related to the α-glucosidase inhibitory effect. MATERIALS AND METHODS: Extracts of a single batch of H. speciosa leaves were prepared by extraction with distinct solvents (ethanol/water in different proportions; methanol/ethyl acetate), employing percolation or static maceration as extraction techniques, at different time intervals. The contents of chlorogenic acid, rutin and FlavHS (a tri-O-glycoside of quercetin) were quantified by a developed and validated HPLC-PDA method. Bornesitol was determined by HPLC-PDA after derivatization with tosyl chloride, whereas total flavonoids were measured spectrophotometrically. Identification of other constituents in the extracts was performed by UPLC-DAD-ESI-MS/MS analysis. The vasorelaxant activity was assayed in rat aortic rings precontracted with phenylephrine, and α-glucosidase inhibition was tested in vitro. Principal component analysis (PCA) was employed to evaluate the contribution of each marker to the biological responses. Isolation of compound 1 was carried out by column chromatography and structure characterization was accomplished by NMR and UPLC-DAD-ESI-MS/MS analyses. RESULTS: The contents of the chemical markers (mean ± s.d. % w/w) varied significantly among the extracts, including total flavonoids (2.68 ± 0.14 to 5.28 ± 0.29), bornesitol (5.11 ± 0.26 to 7.75 ± 0.78), rutin (1.46 ± 0.06 to 1.97 ± 0.02), FlavHS (0.72 ± 0.05 to 0.94 ± 0.14) and chlorogenic acid (0.67 ± 0.09 to 0.91 ± 0.02). All extracts elicited vasorelaxant effect (pIC50 between 4.97 ± 0.22 to 6.48 ± 0.10) and α-glucosidase inhibition (pIC50 between 3.49 ± 0.21 to 4.03 ± 0.10). PCA disclosed positive correlations between the vasorelaxant effect and the contents of chlorogenic acid, rutin, total flavonoids, and FlavHS, whereas a negative correlation was found with bornesitol concentration. No significant correlation between α-glucosidase inhibition and the contents of the above-mentioned compounds was found. On the other hand, PCA carried out with the areas of the ten major peaks from the chromatograms disclosed positive correlations between a peak ascribed to co-eluted triterpenes and α-glucosidase inhibition. A triterpene was isolated and identified as 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol. CONCLUSION: According to PCA results, the vasorelaxant activity of H. speciosa extracts is related to flavonoids and chlorogenic acid, whereas the α-glucosidase inhibition is associated with lipophilic compounds, including esters of lupeol like 3-O-ß-(3'-R-hydroxy)-hexadecanoil-lupeol, described for the first time for the species. These compounds can be selected as chemical markers for the quality control of H. speciosa plant drug and derived extracts.

Ketamine activates the L-arginine/Nitric oxide/cyclic guanosine monophosphate pathway to induce peripheral antinociception in rats.

BACKGROUND: The involvement of the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway in antinociception has been implicated as a molecular mechanism of antinociception produced by several antinociceptive agents, including µ-, κ-, or δ-opioid receptor agonists, nonsteroidal analgesics, cholinergic agonist, and α2C adrenoceptor agonist. In this study, we investigated whether ketamine, a dissociative anesthetic N-methyl-D-aspartate receptor antagonist, was also capable of activating the L-arginine/NO/cGMP pathway and eliciting peripheral antinociception. METHODS: The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2. All drugs were locally administered into the right hindpaw of male Wistar rats. RESULTS: Ketamine (10, 20, 40, 80 µg/paw) elicited a local antinociceptive effect that was antagonized by the nonselective NOS inhibitor L-NOARG (12, 18, and 24 µg/paw) and by the selective neuronal NOS inhibitor L-NPA (12, 18, and 24 µg/paw). In another experiment, we used the inhibitors L-NIO and L-NIL (24 µg/paw) to selectively inhibit endothelial and inducible NOS, respectively. These 2 drugs were ineffective at blocking the effects of the peripheral ketamine injection. In addition, the level of nitrite in the homogenized paw indicated that exogenous ketamine is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ (25, 50, and 100 µg/paw) blocked the action of ketamine, and the cGMP-phosphodiesterase inhibitor zaprinast (50 µg/paw) enhanced the antinociceptive effects of low-dose ketamine (10 µg/paw). CONCLUSIONS: Our results suggest that ketamine stimulates the L-arginine/NO/cyclic GMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.

Polyphenol-rich extract and fractions of Terminalia phaeocarpa Eichler possess hypoglycemic effect, reduce the release of cytokines, and inhibit lipase, α-glucosidase, and α-amilase enzymes.

ETHNOPHARMACOLOGICAL RELEVANCE: species of Terminalia (Combretaceae) are used to treat diabetes and metabolic disorders in Asia, Africa, and America. Terminalia phaeocarpa Eichler is an endemic tree from Brazil, popularly known as capitão. This species is closely related to Terminalia argentea Mart., also vulgarly known as capitão, a native but not endemic tree. Due to their phenotype similarity, these species might eventually prove inseparable and they are indistinctly used by locals to treat diabetes, among other diseases. The potential antidiabetic effect of T. argentea has been previously reported, whereas the biological effects and chemical composition of T. phaeocarpa have never been addressed so far. AIM OF THE STUDY: investigate the hypoglycaemic effect of an ethanol extract (EE) of T. phaeocarpa leaves and its ethyl acetate (FrEtOAc) and hydromethanolic (FrMEOH) fractions, in addition to their activity on the release of pro-inflammatory mediators and inhibition of lipase, α-amylase, and α-glucosidase enzymes. Additionally, it aimed to characterize the chemical composition of the extract and fractions, seeking to identify the compounds related to the biological activities. MATERIALS AND METHODS: The effect on the release of TNF-α, IL-1ß, and CCL-2 was evaluated in LPS-stimulated THP-1 cells (ATCC TIB-202). The inhibition of lipase, α-amylase, and α-glucosidase was tested in vitro, whereas the hypoglycemic effect was assayed in the oral starch tolerance test. The chemical composition was investigated by extensive UHPLC-DAD-ESI-MS/MS analyses. RESULTS: The extract and derived fractions reduced TNF-α (EE pIC50 = 4.58 ± 0.01; FrEtOAc pIC50 = 4.69 ± 0.01; FrMeOH pIC50 = 4.54 ± 0.02) and IL-1ß (EE pIC50 = 4.86 ± 0.02; FrEtOAc pIC50 = 4.86 ± 0.02; FrMeOH pIC50 = 4.75 ± 0.01) release by LPS-stimulated THP-1 cells in a concentration-dependent manner, whereas the inhibitory effect on CCL-2 release did not reach a clear linear relationship for the tested concentrations. The extract and fractions also inhibited in vitro the activity of lipase (EE pIC50 = 3.97 ± 0.12; FrEtOAc pIC50 = 3.87 ± 0.04; FrMeOH pIC50 = 3.67 ± 0.14), α-amylase (EE pIC50 = 4.46 ± 0.27; FrEtOAc pIC50 = 5.47 ± 0.27; FrMeOH pIC50 = 4.26 ± 0.22), and α-glucosidase (EE pIC50 = 5.46 ± 0.05; FrEtOAc pIC50 = 5.79 ± 0.11; FrMeOH pIC50 = 5.74 ± 0.05). The pIC50 values of the test samples were lower than those obtained with orlistat (7.59 ± 0.08) and acarbose (6.04 ± 0.37 and 7.63 ± 0.04) employed as the positive controls respectively in the lipase, α-amylase, and α-glucosidase assays. When assayed in the oral starch tolerance test, the extract and fractions also reduced animal glycaemia. UHPLC-DAD-ESI-MS/MS analyses of the extract and fractions led to the identification of 38 phenolic compounds, mainly phenolic acids, ellagitannins and flavonoids, among others, all of them first-time described for the species. CONCLUSION: Based on our findings, T. phaeocarpa has hypoglycaemic activity and polyphenols are the probable bioactive compounds, which support the ethnomedical use of the species.

Bioguided chemical characterization of pequi (Caryocar brasiliense) fruit peels towards an anti-diabetic activity.

Pequi fruit peels are an underexploited source of polyphenols. The anti-diabetic potential of an extract and fractions from the peels were evaluated in a panel of assays. The extract and fractions thereof inhibited the release of cytokines involved in insulin resistance - TNF, IL-1ß, and CCL2 - by lipopolysaccharide-stimulated THP-1 cells. The ethyl acetate fraction inhibited in vitro α-glucosidase (pIC50 = 4.8 ± 0.1), an enzyme involved in the metabolization of starch and disaccharides to glucose, whereas a fraction enriched in tannins (16C) induced a more potent α-glucosidase inhibition (pIC50 = 5.3 ± 0.1). In the starch tolerance test in mice, fraction 16C reduced blood glucose level (181 ± 10 mg/dL) in comparison to the vehicle-treated group (238 ± 11 mg/dL). UPLC-DAD-ESI-MS/MS analyses disclosed phenolic acids and tannins as constituents, including corilagin and geraniin. These results highlight the potential of pequi fruit peels for developing functional foods to manage type-2 diabetes.

Role of the α7 Nicotinic Acetylcholine Receptor in the Pathophysiology of Atherosclerosis.

Atherosclerosis constitutes a major risk factor for cardiovascular diseases, the leading cause of morbidity and mortality worldwide. This slowly progressing, chronic inflammatory disorder of large- and medium-sized arteries involves complex recruitment of immune cells, lipid accumulation, and vascular structural remodeling. The α7 nicotinic acetylcholine receptor (α7nAChR) is expressed in several cell types involved in the genesis and progression of atherosclerosis, including macrophages, dendritic cells, T and B cells, vascular endothelial and smooth muscle cells (VSMCs). Recently, the α7nAChR has been described as an essential regulator of inflammation as this receptor mediates the inhibition of cytokine synthesis through the cholinergic anti-inflammatory pathway, a mechanism involved in the attenuation of atherosclerotic disease. Aside from the neuronal cholinergic control of inflammation, the non-neuronal cholinergic system similarly regulates the immune function. Acetylcholine released from T cells acts in an autocrine/paracrine fashion at the α7nAChR of various immune cells to modulate immune function. This mechanism additionally has potential implications in reducing atherosclerotic plaque formation. In contrast, the activation of α7nAChR is linked to the induction of angiogenesis and VSMC proliferation, which may contribute to the progression of atherosclerosis. Therefore, both atheroprotective and pro-atherogenic roles are attributed to the stimulation of α7nAChRs, and their role in the genesis and progression of atheromatous plaque is still under debate. This minireview highlights the current knowledge on the involvement of the α7nAChR in the pathophysiology of atherosclerosis.

Determination of l-(+)-bornesitol, the hypotensive constituent of Hancornia speciosa, in rat plasma by LC-MS/MS and its application on a pharmacokinetic study.

Hancornia speciosa is a medicinal plant with proven antihypertensive activity. The cyclitol l-(+)-bornesitol is the main constituent of its leaves and is a potent inhibitor of the angiotensin-converting enzyme. We herein investigated the pharmacokinetic properties of bornesitol administered orally to Wistar rats, as well as bornesitol permeation in Caco-2 cells. Bornesitol was isolated and purified from an ethanol extract of H. speciosa leaves. An ultra-high performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated to quantify bornesitol in rat plasma based on Multiple Reaction Monitoring, using pentaerythritol as an internal standard. Pharmacokinetics was evaluated by the administration of single doses via intravenous in bolus (3 mg/kg) and gavage (3, 15 and 25 mg/kg). Bornesitol permeation was assayed in a transwell Caco-2 cells model, tested alone, or combined with rutin, or as a constituent of H. speciosa extract, using a developed and validated UPLC-ESI-MS/MS method. All assayed validation parameters (selectivity, residual effect, matrix effect, linearity, precision, accuracy and stability of analyte in plasma and solution) for the bioanalytical method met the acceptance criteria established by regulatory guidelines. Bornestiol reached peak plasma concentration within approximately 60 min after oral administration with a half-life ranging from 72.15 min to 123.69 min. The peak concentration and area under the concentration-time curve of bornesitol did not rise proportionally with the increasing doses, suggesting a non-linear pharmacokinetics in rats and the oral bioavailability ranged from 28.5%-59.3%. Bornesitol showed low permeability in Caco-2 cells, but the permeability apparently increased when it was administered either combined with rutin or as a constituent of H. speciosa extract. In conclusion, bornesitol was rapidly absorbed after a single oral administration to rats and followed a non-linear pharmacokinetics. The obtained data will be useful to guide further pre-clinical development of bornesitol-containing herbal preparations of H. speciosa as an antihypertensive agent.

Activation of Cav1.2 and BKCa is involved in the downregulation of caffeine-induced contraction in mice mesenteric arteries.

AIMS: Caffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. MAIN METHODS: Second-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 µM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 µM) and ruthenium red (RuR; 10 µM), transient receptor potential (TRPs) channels blockers; capsazepine (10 µM) and HC067047 (10 µM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 µM), a selective BKCa blocker; and SKF-96365 (30 µM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. KEY FINDINGS: The TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SIGNIFICANCE: Our results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.

Activation of eNOS by D-pinitol Induces an Endothelium-Dependent Vasodilatation in Mouse Mesenteric Artery.

D-pinitol is a cyclitol present in several edible plant species and extensively investigated for the treatment of metabolic diseases in humans, as food supplement, and demonstrated protective effects in the cardiovascular system. For these reasons, the present work aimed at investigating the mechanisms involved in the vascular effects of D-pinitol in mouse mesenteric artery. Mesenteric arteries from male C57BL/6 mice were mounted in a wire myograph. Nitrite was measured by the 2,3-diaminonaphthalene (DAN) method. Protein expression and phosphorylation were measured by Western blot. The systolic blood pressure (SBP) was measured by tail-cuff plethysmography. D-pinitol induced a concentration-dependent vasodilatation in endothelium-intact, but not in endothelium-denuded arteries. Nω-Nitro-L-arginine methyl ester (300 µM) abolished the effect of D-pinitol, while 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM) shifted the concentration-response curve to the right. KN-93 (1 µM) blunted the vasodilator effect of D-pinitol, but H-89 (0.1 µM) did not change it. 1-[2-(Trifluoromethyl) phenyl]imidazole (300 µM), indomethacin (10 µM), celecoxib (5 µM), wortmannin (1 µM), ruthenium red (10 µM), tiron (10 µM), MnTMPyP (30 µM), MPP (0.1 µM), PHTPP (0.1 µM), and atropine (1 µM) did not change the effect of D-pinitol. D-pinitol increased the concentration of nitrite, which was inhibited by L-NAME and calmidazolium (10 µM). D-pinitol increased the phosphorylation level of eNOS activation site at Ser1177 and reduced the phosphorylation level of its inactivation site at Thr495. In normotensive mice, the intraperitoneal administration of D-pinitol (10 mg/kg) induced a significant reduction of the SBP after 30 min. The present results led us to conclude that D-pinitol has an endothelium- and NO-dependent vasodilator effect in mouse mesenteric artery through a mechanism dependent on the activation of eNOS by the calcium-calmodulin complex, which can explain its hypotensive effect in mice.