Arginase II inhibition prevents nitrate tolerance

BACKGROUND AND PURPOSE

Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance.

EXPERIMENTAL APPROACH

Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII –/–) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence.

KEY RESULTS

Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration–response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII –/– mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII –/– mice did not.

CONCLUSION AND IMPLICATIONS

Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.     

Improvement of mechanical heart function by trimetazidine in db/db mice

Aim:

To investigate the influence of trimetazidine, which is known to be an antioxidant and modulator of metabolism, on cardiac function and the development of diabetic cardiomyopathy in db/db mouse.

Methods:

Trimetazidine was administered to db/db mice for eight weeks. Cardiac function was measured by inserting a Millar catheter into the left ventricle, and oxidative stress and AMP-activated protein kinase (AMPK) activity in the myocardium were evaluated.

Results:

Untreated db/db mice exhibited a significant decrease in cardiac function compared to normal C57 mice. Oxidative stress and lipid deposition were markedly increased in the myocardium, concomitant with inactivation of AMPK and increased expression of peroxisome proliferator-activated receptor coactivator-1α (PGC-1α). Trimetazidine significantly improved systolic and diastolic function in hearts of db/db mice and led to reduced production of reactive oxygen species and deposition of fatty acid in cardiomyocytes. Trimetazidine also caused AMPK activation and reduced PGC-1α expression in the hearts of db/db mice.

Conclusion:

The data suggest that trimetazidine significantly improves cardiac function in db/db mice by attenuating lipotoxicity and improving the oxidation status of the heart. Activation of AMPK and decreased expression of PGC-1α were involved in this process. Furthermore, our study suggests that trimetazidine suppresses the development of diabetic cardiomyopathy, which warrants further clinical investigation.     

Preserved arterial vasodilatation via endothelial protease-activated receptor-2 in obese type 2 diabetic mice

BACKGROUND AND PURPOSE

In non-obese diabetic animals, protease-activated receptor-2 (PAR2) agonists are more effective vasodilators, which is attributed to increased COX-2 and endothelial NOS (eNOS) activities. Under conditions of diabetes and obesity, the effectiveness of PAR2 agonists is unknown. We compared the vasodilator responses of small calibre mesenteric arteries from obese diabetic B6.BKS(D)-Lepr^db/J (db/db) induced by PAR2-activating agonists 2-furoyl-LIGRLO-amide (2fly) and trypsin to those obtained in controls [C57BL/6J (C57)], and assessed the contributions of COX, NOS and calcium-activated potassium channels (K_Ca) to these responses.

EXPERIMENTAL APPROACH

Arteries mounted in wire myographs under isometric tension conditions were contracted submaximally by U46619 then exposed to vasodilators. mRNA and protein expression of PAR2, eNOS and soluble GC (sGC) were determined by real-time PCR and Western blots.

KEY RESULTS

ACh- and nitroprusside-induced relaxations were attenuated in db/db compared with C57. In contrast, 2fly- and trypsin-induced relaxations were largely retained in db/db. A NOS inhibitor partly inhibited ACh- and 2fly-induced relaxations in C57, but not those in db/db. Inhibitors of the COX-cAMP pathway ({"type":"entrez-nucleotide","attrs":{"text":"FR122044","term_id":"257958002","term_text":"FR122044"}}FR122044, SC560, NS398, SC58125, SQ22536, CAY10441) did not affect these relaxation responses in either strain. Charybdotoxin (BK_Ca, SK3.1 blocker), but not iberiotoxin (BK_Ca blocker), inhibited responses to the PAR2 agonists in db/db. In db/db protein levels of eNOS were higher, whereas those of sGC were lower than in C57. PAR2 mRNA expression in db/db was higher than in C57.

CONCLUSIONS AND IMPLICATIONS

PAR2-mediated vasodilatation is protected against the negative effects of obesity and diabetes in mice. In diabetic vascular dysfunction, preserved PAR2 vasodilatation was linked to activation of SK3.1.     

Betulinic acid inhibits superoxide anion-mediated impairment of endothelium-dependent relaxation in rat aortas

Objectives:

To investigate the protective effect of betulinic acid (BA) on endothelium-dependent relaxation (EDR) in rat aortas exposed to pyrogallol-produced superoxide anion and its underlying mechanism.

Materials and Methods:

The thoracic aorta of male Sprague-Dawley rats was isolated to mount in the organ bath system and the effect of BA on acetylcholine (ACh)-induced EDR, nitric oxide (NO) level, reactive oxygen species (ROS) level, nitric oxide synthase (NOS) activity, and superoxide dismutase (SOD) activity of aortic rings exposed to pyrogallol (500 μM) for 15 min were measured.

Results:

BA evoked a concentration-dependent EDR in aortas, and pretreatment with EC₅₀ (2.0 μM) concentration of BA markedly enhanced ACh-induced EDR of aortas exposed to pyrogallol-produced superoxide anion (E_max rose from 23.91 ± 5.41% to 42.45 ± 9.99%), which was markedly reversed by both N^w -nitro-L-arginine methyl ester hydrochloride (L-NAME) and methylene blue, but not by indomethacin. Moreover, BA significantly inhibited the increase of ROS level, as well as the decrease of NO level, the endothelial NOS (eNOS) activity, and the SOD activity in aortas induced by pyrogallol-derived superoxide anion.

Conclusion:

These results indicate that BA reduces the impairment of EDR in rat aortas exposed to exogenous superoxide anion, which may closely relate to the reduction of oxidative stress and activation of eNOS–NO pathway.KEY WORDS: Betulinic acid, endothelium-dependent relaxation, nitric oxide oxidative stress, rat thoracic aorta     

Methylglyoxal scavengers attenuate endothelial dysfunction induced by methylglyoxal and high concentrations of glucose

BACKGROUND AND PURPOSE

Endothelial dysfunction is a feature of hypertension and diabetes. Methylglyoxal (MG) is a reactive dicarbonyl metabolite of glucose and its levels are elevated in spontaneously hypertensive rats and in diabetic patients. We investigated if MG induces endothelial dysfunction and whether MG scavengers can prevent endothelial dysfunction induced by MG and high glucose concentrations.

EXPERIMENTAL APPROACH

Endothelium-dependent relaxation was studied in aortic rings from Sprague-Dawley rats. We also used cultured rat aortic and human umbilical vein endothelial cells. The MG was measured by HPLC and Western blotting and assay kits were used.

KEY RESULTS

Incubation of aortic rings with MG (30 µM) or high glucose (25 mM) attenuated endothelium-dependent, acetylcholine-induced relaxation, which was restored by two different MG scavengers, aminoguanidine (100 µM) and N-acetyl cysteine (NAC) (600 µM). Treatment of cultured endothelial cells with MG or high glucose increased cellular MG levels, effects prevented by aminoguanidine and NAC. In cultured endothelial cells, MG and high glucose reduced basal and bradykinin-stimulated nitric oxide (NO) production, cGMP levels, and serine-1177 phosphorylation and activity of endothelial NO synthase (eNOS), without affecting threonine-495 and Akt phosphorylation or total eNOS protein. These effects of MG and high glucose were attenuated by aminoguanidine or NAC.

CONCLUSIONS AND IMPLICATIONS

Our results show for the first time that MG reduced serine-1177 phosphorylation, activity of eNOS and NO production. MG caused endothelial dysfunction similar to that induced by high glucose. Specific and safe MG scavengers have potential to prevent endothelial dysfunction induced by MG and high glucose concentrations.     

Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II-mediated BMP4-oxidative stress cascade

BACKGROUND AND PURPOSE

Boldine is a potent natural antioxidant present in the leaves and bark of the Chilean boldo tree. Here we assessed the protective effects of boldine on endothelium in a range of models of diabetes, ex vivo and in vitro.

EXPERIMENTAL APPROACH

Vascular reactivity was studied in mouse aortas from db/db diabetic and normal mice. Reactive oxygen species (ROS) production, angiotensin AT₁ receptor localization and protein expression of oxidative stress markers in the vascular wall were evaluated by dihydroethidium fluorescence, lucigenin enhanced-chemiluminescence, immunohistochemistry and Western blot respectively. Primary cultures of mouse aortic endothelial cells, exposed to high concentrations of glucose (30 mmol L⁻¹) were also used.

KEY RESULTS

Oral treatment (20 mg kg⁻¹day⁻¹, 7 days) or incubation in vitro with boldine (1 μmol L⁻¹, 12 h) enhanced endothelium-dependent aortic relaxations of db/db mice. Boldine reversed impaired relaxations induced by high glucose or angiotensin II (Ang II) in non-diabetic mouse aortas while it reduced the overproduction of ROS and increased phosphorylation of eNOS in db/db mouse aortas. Elevated expression of oxidative stress markers (bone morphogenic protein 4 (BMP4), nitrotyrosine and AT₁ receptors) were reduced in boldine-treated db/db mouse aortas. Ang II-stimulated BMP4 expression was inhibited by boldine, tempol, noggin or losartan. Boldine inhibited high glucose-stimulated ROS production and restored the decreased phosphorylation of eNOS in mouse aortic endothelial cells in culture.

CONCLUSIONS AND IMPLICATIONS

Boldine reduced oxidative stress and improved endothelium-dependent relaxation in aortas of diabetic mice largely through inhibiting ROS overproduction associated with Ang II-mediated BMP4-dependent mechanisms.     

Impairment of the vascular relaxation and differential expression of caveolin-1 of the aorta of diabetic +db/+db mice

In this study, we compared the endothelium-dependent and -independent relaxation of the isolated thoracic aorta of control (+db/+m) and diabetic (+db/+db) (C57BL/KsJ) mice. The gene expression (mRNA and protein) level of the muscarinic M(3) receptors, endothelial nitric oxide synthase (eNOS) and caveolin-1 of the aorta was also evaluated. Acetylcholine caused a concentration-dependent, N(G)-nitro-L-arginine methyl-ester (20 microM)-sensitive relaxation, with approximately 100% relaxation at 10 microM, in +db/+m mice. In +db/+db mice, the acetylcholine-induced relaxation was significantly smaller (maximum relaxation: approximately 80%). The sodium nitroprusside-mediated relaxation was slightly diminished in +db/+db mice, compared to +db/+m mice. However, there was no significant difference in the isoprenaline- and cromakalim-induced relaxation observed in both species. The mRNA and protein expression levels of caveolin-1 were significantly higher in the aorta of +db/+db mice. In contrast, there was no difference in the mRNA and protein expression levels of eNOS and muscarinic M(3) receptors between these mice. Our results demonstrate that the impairment of the acetylcholine-induced, endothelium-dependent aortic relaxation observed in +db/+db mice was probably associated with an enhanced expression of caveolin-1 mRNA and protein.     

Effects of aldosterone and related steroids on LPS-induced increased expression of inducible NOS in rat aortic smooth muscle cells

BACKGROUND AND PURPOSE

Expression of inducible NOS (iNOS) is important in certain inflammatory diseases. We determined if the hormone aldosterone, a mineralocorticoid receptor (MR) agonist, affects LPS activation of iNOS expression in rat aortic smooth muscle cells (RASMC).

EXPERIMENTAL APPROACH

Cultured RASMC were treated with LPS, with or without agonists/antagonists of steroid receptors. iNOS expression was determined by nitrite assays on culture medium removed from treated cells and by immunoblotting of cell protein extracts.

KEY RESULTS

LPS (1 µg·mL⁻¹) increased nitrite and iNOS protein above that in control (untreated) cells. These effects of LPS were reduced by aldosterone (0.1–10 µM). The MR antagonists, eplerenone (10 µM) and spironolactone (10 or 50 µM), did not inhibit these actions of 1 µM aldosterone, but the latter were prevented by 10 µM mifepristone, a glucocorticoid (GR) and progestogen receptor (PR) antagonist. Mifepristone also prevented the reduction of LPS-induced nitrite increase produced by 1 µM dexamethasone (GR agonist) and 10 µM progesterone (PR agonist). Spironolactone (10–50 µM) by itself decreased LPS-induced increases in nitrite and iNOS protein. Mifepristone (10 µM) partially reversed these effects of 10 µM spironolactone, but not those of 50 µM; the effects of 50 µM spironolactone were also unchanged when mifepristone was increased to 50 µM.

CONCLUSIONS AND IMPLICATIONS

This pharmacological profile suggests that aldosterone, and possibly 10 µM spironolactone, use mechanisms that are dependent on PR and/or GR, but not MR, to inhibit iNOS induction in RASMC. With 50 µM spironolactone, other inhibitory mechanisms requiring further investigation may become predominant.     

Inhibition of PKC-θ preserves cardiac function and reduces fibrosis in streptozotocin-induced diabetic cardiomyopathy

Background and Purpose

T-cell infiltration, interstitial fibrosis and cardiac dysfunction have been observed in diabetic patients with cardiovascular diseases. PKC-θ is crucial for the activation of mature T-cells. We hypothesized that inhibition of PKC-θ might protect diabetic hearts through inhibition of T-cell stimulation and maintenance of tight junction integrity.

Experimental Approach

A model of type 1 diabetes was induced by streptozotocin (STZ) (50 mg kg^–1 for 5 days) in male C57BL/6J wild-type (WT) mice and Rag1 knockout (KO) mice which lack mature lymphocytes. A cell-permeable selective PKC-θ peptide inhibitor (PI) was administered i.p. (0.2 mg kg^–1·day^–1) for 4 weeks (first phase) and 2 weeks (second phase). At the end of the 11th week, cardiac contractile force was measured in isolated perfused hearts. Cardiac morphology and fibrosis were determined. Phosphorylation of PKC-θ at Tyr³⁵⁸, infiltrated T-cells and tight junction protein ZO-1 within the hearts were detected, using immunohistochemcial techniques.

Key Results

PI did not affect high blood glucose level in both WT and Rag1 KO diabetic mice. Diabetes induced cardiac fibrosis in WT mice but not in Rag1 KO mice. PI attenuated cardiac fibrosis and improved cardiac contractility of WT diabetic hearts. PI decreased expression of phosphorylated PKC-θ, reduced the infiltration of T-cells and increased ZO-1 expression within WT diabetic hearts.

Conclusion and Implications

Inhibition of PKC-θ improves cardiac function and reduces cardiac fibrosis in WT mice with streptozotocin-induced diabetes. Mature T-cells play a key role in pathophysiology of diabetic cardiomyopathy.     

The xanthine derivative KMUP-1 inhibits models of pulmonary artery hypertension via increased NO and cGMP-dependent inhibition of RhoA/Rho kinase

Background and purpose:

KMUP-1 is known to increase cGMP, enhance endothelial nitric oxide synthase (eNOS) and suppress Rho kinase (ROCK) expression in smooth muscle. Here, we investigated the mechanism of action of KMUP-1 on acute and chronic pulmonary artery hypertension (PAH) in rats.

Experimental approach:

We measured pulmonary vascular contractility, wall thickening, eNOS immunostaining, expressions of ROCK II, RhoA activation, myosin phosphatase target subunit 1 (MYPT1) phosphorylation, eNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG) and phosphodiesterase 5A (PDE-5A), blood oxygenation and cGMP/cAMP, and right ventricular hypertrophy (RVH) in rats.

Key results:

In rings of intact pulmonary artery (PA), KMUP-1 relaxed the vasoconstriction induced by phenylephrine (10 µM) or the thromboxane A₂-mimetic U46619 (0.5 µM). In endothelium-denuded PA rings, this relaxation was reduced. In acute PAH induced by U46619 (2.5 µg·kg⁻¹·min⁻¹, 30 min), KMUP-1 relaxed vasoconstriction by enhancing levels of eNOS, sGC and PKG, suppressing those of PDE-5A, RhoA/ROCK II activation and MYPT1 phosphorylation, and restoring oxygenation in blood and cGMP/cAMP in plasma. Incubating smooth muscle cells from PA (PASMCs) with KMUP-1 inhibited thapsigargin-induced Ca²⁺ efflux and angiotensin II-induced Ca²⁺ influx. In chronic PAH model induced by monocrotaline, KMUP-1 increased eNOS and reduced RhoA/ROCK II activation/expression, PA wall thickening, eNOS immunostaining and RVH. KMUP-1 and sildenafil did not inhibit monocrotaline-induced PDE-5A expression.

Conclusion and implications:

KMUP-1 decreased PAH by enhancing NO synthesis by eNOS, with consequent cGMP-dependent inhibition of RhoA/ROCK II and Ca²⁺ desensitization in PASMCs. KMUP-1 has the potential to reduce vascular resistance, remodelling and RVH in PAH.     

Effects of pyrazole partial agonists on HCA(2) -mediated flushing and VLDL-triglyceride levels in mice

BACKGROUND AND PURPOSE

Niacin can effectively treat dyslipidaemic disorders. However, its clinical use is limited due to the cutaneous flushing mediated by the nicotinic acid receptor HCA₂. In the current study, we evaluated two partial agonists for HCA₂, LUF6281 and LUF6283, with respect to their anti-dyslipidaemic potential and cutaneous flushing effect.

EXPERIMENTAL APPROACH

In vitro potency and efficacy studies with niacin and the two HCA₂ partial agonists were performed using HEK293T cells stably expressing human HCA₂. Normolipidaemic C57BL/6 mice received either niacin or the HCA₂ partial agonists (400 mg·kg⁻¹·day⁻¹) once a day for 4 weeks for evaluation of their effects in vivo.

KEY RESULTS

Radioligand competitive binding assay showed K_i values for LUF6281 and LUF6283 of 3 and 0.55 µM. [³⁵S]-GTPγS binding revealed the rank order of their potency as niacin > LUF6283 > LUF6281. All three compounds reduced plasma VLDL-triglyceride concentrations similarly, while LUF6281 and LUF6283, in contrast to niacin, did not also exhibit the unwanted flushing side effect in C57BL/6 mice. Niacin reduced the expression of lipolytic genes HSL and ATGL in adipose tissue by 50%, whereas LUF6281 and LUF6283 unexpectedly did not. In contrast, the decrease in VLDL-triglyceride concentration induced by LUF6281 and LUF6283 was associated with a parallel >40% reduced expression of APOB within the liver.

CONCLUSIONS AND IMPLICATIONS

The current study identifies LUF6281 and LUF6283, two HCA₂ partial agonists of the pyrazole class, as promising drug candidates to achieve the beneficial lipid lowering effect of niacin without producing the unwanted flushing side effect.     

Combining a dipeptidyl peptidase-4 inhibitor, alogliptin, with pioglitazone improves glycaemic control, lipid profiles and ��-cell function in db/db mice

Background and purpose:

Alogliptin, a highly selective dipeptidyl peptidase-4 (DPP-4) inhibitor, enhances incretin action and pioglitazone enhances hepatic and peripheral insulin actions. Here, we have evaluated the effects of combining these agents in diabetic mice.

Experimental approach:

Effects of short-term treatment with alogliptin alone (0.01%–0.1% in diet), and chronic combination treatment with alogliptin (0.03% in diet) and pioglitazone (0.0075% in diet) were evaluated in db/db mice exhibiting early stages of diabetes.

Key results:

Alogliptin inhibited plasma DPP-4 activity up to 84% and increased plasma active glucagon-like peptide-1 by 4.4- to 4.9-fold. Unexpectedly, alogliptin alone lacked clear efficacy for improving glucose levels. However, alogliptin in combination with pioglitazone clearly enhanced the effects of pioglitazone alone. After 3–4 weeks of treatment, combination treatment increased plasma insulin by 3.8-fold, decreased plasma glucagon by 41%, both of which were greater than each drug alone, and increased plasma adiponectin by 2.4-fold. In addition, combination treatment decreased glycosylated haemoglobin by 2.2%, plasma glucose by 52%, plasma triglycerides by 77% and non-esterified fatty acids by 48%, all of which were greater than each drug alone. Combination treatment also increased expression of insulin and pancreatic and duodenal homeobox 1 (PDX1), maintained normal β-cell/α-cell distribution in islets and restored pancreatic insulin content to levels comparable to non-diabetic mice.

Conclusions and implications:

These results indicate that combination treatment with alogliptin and pioglitazone at an early stage of diabetes improved metabolic profiles and indices that measure β-cell function, and maintained islet structure in db/db mice, compared with either alogliptin or pioglitazone monotherapy.     

Bradykinin and B₂ receptor antagonism in rat and human articular chondrocytes

BACKGROUND AND PURPOSE

In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis.

EXPERIMENTAL APPROACH

BK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8.

KEY RESULTS

Density of [³H]-BK binding sites was higher (13–30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B₂ receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B₁ receptor antagonist Lys-[Leu⁸][desArg⁹]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798 196, 200 nM; L-161 982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-κB.

CONCLUSION AND IMPLICATIONS

BK mediated inflammatory changes and cartilage degradation and B₂ receptor blockade would, therefore, be a potential treatment for OA.     

The intestinal anti-inflammatory effect of dersalazine sodium is related to a down-regulation in IL-17 production in experimental models of rodent colitis

BACKGROUND AND PURPOSE

Dersalazine sodium (DS) is a new chemical entity formed by combining, through an azo bond, a potent platelet activating factor (PAF) antagonist (UR-12715) with 5-aminosalicylic acid (5-ASA). DS has been demonstrated to have anti-inflammatory effects on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats and recently in UC patients in phase II PoC. There is Increasing evidence that Th17 cells have an important role in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to further characterize the anti-inflammatory effects of DS.

EXPERIMENTAL APPROACH

Effect of DS (10 or 30 mg·kg⁻¹ b.i.d.) on TNBS-induced colitis in rats was studied after 2 and 7 days with special focus on inflammatory mediators. Additionally, its anti-inflammatory properties were analysed in two different models of dextran sodium sulphate (DSS)-induced colitis, BALB/c and C57BL/6 mice, the latter being dependent on IL-17.

KEY RESULTS

DS, when administered for 7 days, showed intestinal anti-inflammatory effects in TNBS-induced colitis; these effects were observed both macroscopically and through the profile of inflammatory mediators (TNF, IL-1β, IL-6 and IL-17). Although the 2 day treatment with DS did not induce intestinal anti-inflammatory effects, it was sufficient to reduce the enhanced IL-17 expression. DS showed beneficial effects on DSS-induced colitis in C57BL/6 mice and reduced colonic pro-inflammatory cytokines IL-1β, IL-6 and IL-17. In contrast, it did not exert intestinal anti-inflammatory effects on DSS-induced colitis in BALB/c mice.

CONCLUSIONS AND IMPLICATIONS

DS exerts intestinal anti-inflammatory activity in different rodent models of colitis through down-regulation of IL-17 expression.     

Chronic treatment with angiotensin-(1-7) improves renal endothelial dysfunction in apolipoproteinE-deficient mice

BACKGROUND AND PURPOSE

ApolipoproteinE-deficient [apoE (−/−)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin-(1-7) [Ang-(1-7)], acting through its specific GPCR, the Mas receptor, has endothelium-dependent vasodilator properties. Here we have investigated if chronic treatment with Ang-(1-7) improved endothelial dysfunction in apoE (−/−) mice.

EXPERIMENTAL APPROACH

ApoE (−/−) mice fed on a lipid-rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang-(1-7) (82 µg·kg⁻¹·h⁻¹) or the same dose of Ang-(1-7) together with D-Ala-Ang-(1-7) (125 µg·kg⁻¹·h⁻¹) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys.

KEY RESULTS

Ang-(1-7)-treated apoE (−/−) mice showed improved renal endothelium-dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (−/−) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium-dependent vasorelaxation in kidneys of saline-treated apoE (−/−) mice whereas no effect was observed in Ang-(1-7)-treated mice. Chronic treatment with D-Ala-Ang-(1-7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang-(1-7) on endothelium-dependent vasorelaxation. Renal endothelium-independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang-(1-7)-treated mice, compared with untreated mice, whereas eNOS expression was increased.

CONCLUSION AND IMPLICATIONS

Chronic infusion of Ang-(1-7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.     

5α-reduced glucocorticoids exhibit dissociated anti-inflammatory and metabolic effects

BACKGROUND AND PURPOSE

Dissociating anti-inflammatory efficacy from the metabolic side effects of glucocorticoids is an attractive therapeutic goal. 5α-Tetrahydro-corticosterone (5αTHB), produced from corticosterone by 5α-reductases, activates glucocorticoid receptors. This study compares the effects of 5αTHB on inflammation and metabolism in vitro and in vivo.

METHODS

Suppression of cytokine release by 5αTHB and corticosterone were studied following LPS activation of mouse bone marrow derived macrophages. In vivo the efficacy of these steroids to dysregulate metabolic homeostasis and modulate immune suppression and the responses to thioglycollate-induced peritonitis in C57BL/6 mice were studied following acute injection (1.5–15 mg) and chronic infusion (50 µg·day⁻¹, 14 days).

RESULTS

In macrophages, 5αTHB increased secretion of IL-10 similarly to corticosterone (180%, 340%; data are % vehicle, treated with 5αTHB and corticosterone, respectively) and suppressed LPS-induced secretion of TNF-α (21.9%, 74.2%) and IL-6 (16.4%, 69.4%). In mice with thioglycollate-induced peritonitis, both 5αTHB and corticosterone reduced the numbers of neutrophils (58.6%, 49.9%) and inflammatory monocytes (69.5%, 96.4%), and also suppressed MCP-1 (48.7%, 80.9%) and IL-6 (53.5%, 86.7%) in peritoneal exudate. In mice chronically infused with 5αTHB and corticosterone LPS-induced production of TNF-α from whole blood was suppressed to the same degree (63.2%, 37.2%). However, in contrast to corticosterone, 5αTHB did not induce body weight loss, increase blood pressure or induce hyperinsulinaemia.

CONCLUSIONS

5αTHB has anti-inflammatory effects in vitro and in vivo. At doses with equivalent anti-inflammatory efficacy to corticosterone, 5αTHB did not induce metabolic toxicity and thus may be a prototype for a safer anti-inflammatory drug.     

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

BACKGROUND AND PURPOSE

To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.

EXPERIMENTAL APPROACH

Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs.

KEY RESULTS

18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.

CONCLUSION AND IMPLICATIONS

When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.