Grazielle C. Silva

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 &mgr;-, &kgr;-, or &dgr;-opioid receptor agonists, nonsteroidal analgesics, cholinergic agonist, and &agr;2C 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 &mgr;g/paw) elicited a local antinociceptive effect that was antagonized by the nonselective NOS inhibitor l-NOARG (12, 18, and 24 &mgr;g/paw) and by the selective neuronal NOS inhibitor l-NPA (12, 18, and 24 &mgr;g/paw). In another experiment, we used the inhibitors l-NIO and l-NIL (24 &mgr;g/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 &mgr;g/paw) blocked the action of ketamine, and the cGMP-phosphodiesterase inhibitor zaprinast (50 &mgr;g/paw) enhanced the antinociceptive effects of low-dose ketamine (10 &mgr;g/paw). CONCLUSIONS: Our results suggest that ketamine stimulates the l-arginine/NO/cyclic GMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.

Hancornia speciosa Gomes induces hypotensive effect through inhibition of ACE and increase on NO.

ETHNOPHARMACOLOGICAL RELEVANCE The leaves of Hancornia speciosa Gomes are popularly used in Brazil to treat diabetes and hypertension. Cardiovascular diseases are the main cause of death worldwide and their incidences are increasing in Brazilian population. The present study aimed to investigate the hypotensive effect and the mechanism of action of Hancornia speciosa Gomes. METHODS A fraction of the ethanolic extract of leaves from Hancornia speciosa (SFH) was obtained and standardized by its content on rutin, bornesitol and quinic acid. Systolic blood pressure (SBP) of normotensive mice was measured by tail plethysmography. SFH was given orally and SBP was monitored for 5h. Angiotensin-converting enzyme (ACE) inhibitor activity of SFH (1mg/kg) or captopril (10mg/kg) was measured by colorimetric methods. Serum nitrite levels were measured by spectrophotometry. RESULTS SFH induced a dose-dependent hypotensive effect in normotensive mice. The serum activity of ACE and the level of angiotensin II were significantly reduced by SFH and by captopril. Administration of SFH induced a significant increase on plasmatic level of nitrites and the systemic inhibition of nitric oxide synthase by L-NAME (20mg/kg) reduced the hypotensive effect of SFH. CONCLUSIONS The present work demonstrated that Hancornia speciosa has a potent hypotensive effect in normotensive mice. The inhibition of ACE leading to reduction on angiotensin II and increase on NO levels might account for the hypotensive effect. These results support the use of Hancornia speciosa by traditional medicine as antihypertensive.

The Nitric oxide/CGMP/KATP pathway mediates systemic and central antinociception induced by resistance exercise in rats.

Resistance exercise (RE) is characterized to increase strength, tone, mass, and/or muscular endurance and also for produces many beneficial effects, such as blood pressure and osteoporosis reduction, diabetes mellitus control, and analgesia. However, few studies have investigated endogenous mechanisms involved in the RE-induced analgesia. Thus, the aim of this study was evaluate the role of the NO/CGMP/KATP pathway in the antinociception induced by RE. Wistar rats were submitted to acute RE in a weight-lifting model. The nociceptive threshold was measured by mechanical nociceptive test (paw-withdrawal). To investigate the involvement of the NO/CGMP/KATP pathway the following nitric oxide synthase (NOS) non-specific and specific inhibitors were used: N-nitro-l-arginine (NOArg), Aminoguanidine, N5-(1-Iminoethyl)-l-ornithine dihydrocloride (l-NIO), Nω-Propyl-l-arginine (l-NPA); guanylyl cyclase inhibitor, 1H-[1,2,4]oxidiazolo[4,3-a]quinoxalin-1-one (ODQ); and KATP channel blocker, Glybenclamide; all administered subcutaneously, intrathecally and intracerebroventricularly. Plasma and cerebrospinal fluid (CSF) nitrite levels were determined by spectrophotometry. The RE protocol produced antinociception, which was significantly reversed by NOS specific and unspecific inhibitors, guanylyl cyclase inhibitor (ODQ) and KATP channel blocker (Glybenclamide). RE was also responsible for increasing nitrite levels in both plasma and CSF. These finding suggest that the NO/CGMP/KATP pathway participates in antinociception induced by RE.

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 Ca²⁺-free solution showed that tiliroside antagonized CaCl₂-induced contractions. Moreover, tiliroside reduced the rise in intracellular Ca²⁺ concentration induced by membrane depolarization in vascular smooth muscle cells. Finally, tiliroside decreased the voltage-activated peak amplitude of the L-type Ca²⁺ 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 Ca²⁺ channel in smooth muscle cells.

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 CB2 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, α2C‐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 E2. 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.

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.

Ang-(1-7) activates the NO/cGMP and ATP-sensitive K+ channels pathway to induce peripheral antinociception in rats.

Angiotensin-(1-7) is a bioactive component of the renin-angiotensin system that is formed endogenously and induces nitric oxide release in several tissues. The L-arginine/NO/cyclic GMP pathway and ATP-sensitive K+ channels have been proposed as the mechanism of action for the peripheral antinociception of several groups of drug and endogenous substances, including opioids, non-steroidal analgesics, acetylcholine and others. The aim of the present study was to investigate the involvement of the L-arginine/NO/cGMP and KATP+ pathway on antinociception induced by angiotensin-(1-7). Paw pressure in rats was used to induce hyperalgesia via an intraplantar injection of prostaglandin E2 (2 μg/paw). Ang-(1-7) (2, 3 and 4 μg/paw) 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. The selective inhibition of endothelial (eNOS) and inducible (iNOS) NOS by L-NIO and L-NIL, respectively, was ineffective at blocking the effects of a local Ang-(1-7) injection. In addition, the level of nitrite in the homogenized paw tissue, as determined by a colorimetric assay, indicated that exogenous Ang-(1-7) is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ and the specific blocker of ATP-sensitive K+ channels glibenclamide (40, 80 and 160 μg/paw) antagonized the Ang-(1-7) response. The results provide evidence that Ang-(1-7) most likely induces peripheral antinociceptive effects via the L-arginine/NO/cGMP pathway and KATP+ pathway activation.

ACE inhibition by astilbin isolated from Erythroxylum gonocladum (Mart.) O.E. Schulz.

Erythroxylum species have several traditional uses in different countries, including the treatment of hypertension. The ethanol extract from E. gonocladum aerial parts, a species endemic to the Brazilian cerrado, elicited a concentration-dependent inhibition of angiotensin converting enzyme (ACE) (pIC(50)=4.53+/-0.05). Extract fractionation led to the isolation of two compounds, whose structures were assigned by spectrometric data as astilbin and beta-sitosterol, along with a mixture of palmitic, stearic and linolenic acids. This is the first report on the occurrence of these compounds on E. gonocladum. Astilbin promoted significant ACE inhibition in vitro (pIC(50)=5.86+/-0.33) and its activity did not differ from captopril, when both compounds were assayed at 10 microM concentration.

Potent antihypertensive effect of Hancornia speciosa leaves extract

BACKGROUND Hancornia speciosa Gomes is an herb traditionally used in Brazil for blood pressure control. PURPOSE The present work investigated the antihypertensive effect of an extract from Hancornia speciosa leaves (SFH) and analyzed its underlying mechanisms of action. METHODS Hypertension was induced in mice by surgical removal of a kidney and by subcutaneous administration of a pellet with deoxycorticosterone. Vasodilatation was measured in mesenteric arteries with a wire myograph. Nitrites were measured by fluorescence with 2,3-diaminonaphthalene and H2O2 was measured with carbon microsensors. RESULTS SFH (0.03, 0.1 or 1 mg/kg; po) induced a dose-dependent, long-lasting reduction in the systolic blood pressure in conscious DOCA-salt hypertensive mice (DOCA). Administration of SFH produced a significant increase in the plasmatic level of nitrites. The systemic inhibition of nitric oxide synthase by L-NAME (20 mg/kg) reduced its antihypertensive effect. SFH also induced a concentration-dependent vasodilatation of mesenteric resistance arteries contracted with phenylephrine, which was more potent in arteries from DOCA mice. Removal of the endothelium or pretreatment with L-NAME or catalase reduced the vasodilator response for SFH. The nitrite production induced by SFH was significantly bigger in mesenteric arteries from DOCA than in SHAM mice. However, the production of H2O2 induced by SFH was twice higher in DOCA mice. CONCLUSION Altogether, our results point to an antihypertensive effect of SFH due to a reduction in peripheral resistance through the production of NO and by a mechanism involving an increased production of H2O2 in the mesenteric arteries from hypertensive mice. These findings are further evidence to support the use of Hancornia speciosa by traditional medicine as an antihypertensive drug.

Effects of sepsis-induced acute lung injury on glycogen content in different tissues

ABSTRACT The metabolic profile is very affected in sepsis, which is the most important cause of extrapulmonary acute lung injury (ALI-EX). The aim of the present study was to investigate whether sepsis-induced ALI-EX in mice affects the glycogen content in different tissues. This measurement could indicate performance limitations of tissues and constitute a novel biochemical aspect of ALI. ALI was induced by cecal ligation and puncture (CLP), which is a model that reproduces clinical and pathological alterations stemming from sepsis. Control group mice were sham-operated. Glycogen content (mg/g tissue) from different tissues was measured using the anthrone reagent. Glycogen content in the diaphragm (0.3 ± 0.1) and gastrocnemius muscle (0.4 ± 0.1) was lower in the sepsis group than the control group (0.9 ± 0.1 and 1.1 ± 0.2, respectively). However, there were no significant differences in glycogen content in the heart and kidney. Sepsis caused a greater thickening of the alveolar walls, more areas of atelectasis, and a greater abundance of inflammatory cells in comparison to the control group. These results demonstrate that glycogen content in sepsis-induced ALI-EX is altered in different tissues.