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.     

Arginase inhibition alleviates hypertension in the metabolic syndrome

Background and Purpose

We have previously shown that arginase inhibition alleviates hypertension associated with in a diabetic animal model. Here, we investigated the protective effect of arginase inhibition on hypertension in metabolic syndrome.

Experimental Approach

Metabolic syndrome was induced in rats by administration of fructose (10% in drinking water) for 12 weeks to induce vascular dysfunction. Three arginase inhibitors (citrulline, norvaline and ornithine) were administered daily in the last 6 weeks of study before and tail BP was recorded in conscious animals. Concentration response curves for phenylephrine (PE), KCl and ACh in addition to ACh-induced NO generation were obtained in thoracic aorta rings. Serum glucose, insulin, uric acid and lipid profile were determined as well as reactive oxygen species (ROS) and arginase activity.

Key Results

Arginase activity was elevated in metabolic syndrome while significantly inhibited by citrulline, norvaline or ornithine treatment. Metabolic syndrome was associated with elevations in systolic and diastolic BP, while arginase inhibition significantly reduced elevations in diastolic and systolic BP. Metabolic syndrome increased vasoconstriction responses of aorta to PE and KCl and decreased vasorelaxation to ACh, while arginase inhibition completely prevented impaired responses to ACh. In addition, arginase inhibition prevented impaired NO generation and exaggerated ROS formation in metabolic syndrome. Furthermore, arginase inhibition significantly reduced hyperinsulinaemia and hypertriglyceridaemia without affecting hyperuricaemia or hypercholesterolaemia associated with metabolic syndrome.

Conclusions and Implications

Arginase inhibition alleviates hypertension in metabolic syndrome directly through endothelial-dependent relaxation/NO signalling protection and indirectly through inhibition of insulin resistance and hypertriglyceridaemia.     

Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism

AIMS

Oral L-arginine supplementation has been used in several studies to improve endothelium-dependent, nitric oxide (NO)-mediated vasodilation. L-Arginine treatment is hampered by extensive presystemic elimination due to intestinal arginase activity. In contrast, L-citrulline is readily absorbed and at least in part converted to L-arginine. The aim of our study was to assess this metabolic conversion and its subsequent pharmacodynamic effects.

METHODS

In a double-blind, randomized, placebo-controlled cross-over study, 20 healthy volunteers received six different dosing regimes of placebo, citrulline, and arginine. Pharmacokinetic parameters (C_max, T_max, C_min, AUC) were calculated after 1 week of oral supplementation. The ratio of plasma L-arginine over asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase (arginine/ADMA ratio), urinary cyclic guanosine monophosphate (cGMP) and nitrate excretion rates, and flow-mediated vasodilation (FMD) was measured to assess pharmacodynamic effects.

RESULTS

L-Citrulline dose-dependently increased AUC and C_max of plasma L-arginine concentration more effectively than L-arginine (P < 0.01). The highest dose of citrulline (3 g bid) increased the C_min of plasma L-arginine and improved the L-arginine/ADMA ratio from 186 ± 8 (baseline) to 278 ± 14 [P < 0.01, 95% confidence interval (CI) 66, 121]. Moreover, urinary nitrate and cGMP were increased from 92 ± 10 to 125 ± 15 µmol mmol⁻¹ creatinine (P = 0.01, 95% CI 8, 58) and from 38 ± 3.3 to 50 ± 6.7 nmol mmol⁻¹ creatinine (P = 0.04, 95% CI 0.4, 24), respectively. No treatment improved FMD over baseline. However, pooled analysis of all FMD data revealed a correlation between the increase of arginine/ADMA ratio and improvement of FMD.

CONCLUSION

Our data show for the first time that oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling in a dose-dependent manner.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• L-Arginine is a semiessential amino acid that is converted to nitric oxide (NO) by NO synthase (NOS).
• NO improves endothelial function by elevating cyclic guanosine monophosphate.
• However, oral L-arginine treatment in humans is hampered by extensive metabolism.

WHAT THIS STUDY ADDS

• Oral L-citrulline supplementation raises plasma L-arginine concentration and augments NO-dependent signalling in a dose-dependent manner.
• L-Citrulline may thus be an alternative to L-arginine in patients with impaired NOS activity.

     

Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

Background and Purpose

Emerging evidence suggests a selective up-regulation of arginase I in diabetes causing coronary artery disease; however, the mechanisms behind this up-regulation are still unknown. Activated p38 MAPK has been reported to increase arginase II in various cardiovascular diseases. We therefore tested the role of p38 MAPK in the regulation of arginase I and II expression and its effect on endothelial dysfunction in diabetes mellitus.

Experimental Approach

Endothelial function was determined in septal coronary (SCA), left anterior descending coronary (LAD) and mesenteric (MA) arteries from healthy and streptozotocin-induced diabetic Wistar rats by wire myographs. Arginase activity and protein levels of arginase I, II, phospho-p38 MAPK and phospho-endothelial NOS (eNOS) (Ser¹¹⁷⁷) were determined in these arteries from diabetic and healthy rats treated with a p38 MAPK inhibitor in vivo.

Key Results

Diabetic SCA and MA displayed impaired endothelium-dependent relaxation, which was prevented by arginase and p38 MAPK inhibition while LAD relaxation was not affected. Arginase I, phospho-p38 MAPK and eNOS protein expression was increased in diabetic coronary arteries. In diabetic MA, however, increased expression of arginase II and phospho-p38 MAPK, increased arginase activity and decreased expression of eNOS were observed. All these effects were reversed by p38 MAPK inhibition.

Conclusions and Implications

Diabetes-induced activation of p38 MAPK causes endothelial dysfunction via selective up-regulation of arginase I expression in coronary arteries and arginase II expression in MA. Therefore, regional differences appear to exist in the arginase isoforms contributing to endothelial dysfunction in type 1 diabetes mellitus.     

A new class of organic nitrates: investigations on bioactivation,tolerance and cross-tolerance phenomena

Background and purpose:

The chronic use of organic nitrates is limited by serious side effects including oxidative stress, nitrate tolerance and/or endothelial dysfunction. The side effects and potency of nitroglycerine depend on mitochondrial aldehyde dehydrogenase (ALDH-2). We sought to determine whether this concept can be extended to a new class of organic nitrates with amino moieties (aminoalkyl nitrates).

Experimental approach:

Vasodilator potency of the organic nitrates, in vitro tolerance and in vivo tolerance (after continuous infusion for 3 days) were assessed in wild-type and ALDH-2 knockout mice by isometric tension studies. Mitochondrial oxidative stress was analysed by L-012-dependent chemiluminescence and protein tyrosine nitration.

Key results:

Aminoethyl nitrate (AEN) showed an almost similar potency to glyceryl trinitrate (GTN), even though it is only a mononitrate. AEN-dependent vasodilatation was mediated by cGMP and nitric oxide. In contrast to triethanolamine trinitrate (TEAN) and GTN, AEN bioactivation did not depend on ALDH-2 and caused no in vitro tolerance. In vivo treatment with TEAN and GTN, but not with AEN, induced cross-tolerance to acetylcholine (ACh)-dependent and GTN-dependent relaxation. Although all nitrates tested induced tolerance to themselves, only TEAN and GTN significantly increased mitochondrial oxidative stress in vitro and in vivo.

Conclusions and implications:

The present results demonstrate that not all high potency nitrates are bioactivated by ALDH-2 and that high potency of a given nitrate is not necessarily associated with induction of oxidative stress or nitrate tolerance. Obviously, there are distinct pathways for bioactivation of organic nitrates, which for AEN may involve xanthine oxidoreductase rather than P450 enzymes.     

Investigating the interaction of McN-A-343 with the M1 muscarinic receptor using its nitrogen mustard derivative and ACh mustard

Background and purpose:

We investigated how McN-A-343 inhibited the alkylation of the M₁ muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically.

Experimental approach:

We incubated the M₁ muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [³H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model.

Key results:

The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [³H]N-methylscopolamine binding.

Conclusions and implications:

Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M₁ muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.     

Cyclo-oxygenase-2 inhibition and endothelium-dependent vasodilation in younger vs. older healthy adults

Aim

A major feature of endothelial dysfunction is reduced endothelium-dependent vasodilation, which in ageing may be due to decreased production of endothelial prostacyclin, or nitric oxide (NO), or both.

Method

We tested this hypothesis in 12 younger (age 18–38 years, six women) and 12 older healthy adults (age 55–73 years, six post-menopausal women). Endothelium-dependent vasodilation was assessed by the forearm vascular conductance (FVC) response to intra-arterial acetylcholine (ACh) (0.5, 1.0, 2.0, 4.0 μg dl⁻¹ forearm tissue min⁻¹) before and 90 min after inhibition of the enzyme cyclo-oxygenase-2 (COX-2) with oral celecoxib (400 mg), followed by the addition of endothelial NO synthase inhibition with intra-arterial N^G-monomethyl-l arginine acetate (L-NMMA).

Results

Ageing was associated with a significantly reduced FVC response to ACh (P = 0.009, age-by-dose interaction; highest dose FVC ± SEM in ageing: 11.2 ± 1.4 vs. younger: 17.7 ± 2.4 units, P = 0.02). Celecoxib did not reduce resting FVC or the responses to ACh in any group. L-NMMA significantly reduced resting FVC and the responses to ACh in all groups, and absolute FVC values following L-NMMA were similar between groups.

Conclusion

In healthy normotensive younger and older adults, there is minimal contribution of prostacyclin to ACh-mediated vasodilation, yet the NO component of vasodilation is reduced with ageing. In the clinical context, these findings suggest that acute administration of medications that inhibit prostacyclin (i.e. COX-2 inhibitors) evoke modest vascular consequences in healthy persons. Additional studies are necessary to test whether chronic use of COX-2 medications reduces endothelium dependent vasodilation in older persons with or without cardiovascular risk factors.     

Galantamine improves apomorphine-induced deficits in prepulse inhibition via muscarinic ACh receptors in mice

Background and purpose

Galantamine, a weak acetylcholine esterase (AChE) inhibitor and allosteric potentiator of nicotinic ACh receptors (nAChRs), improves apomorphine-induced deficits in prepulse inhibition (PPI), sensory information-processing deficits, via a nAChR-independent mechanism. The present study examined the role of muscarinic ACh receptors (mAChRs) in the effect of galantamine, and studied the mechanism of galantamine-induced increases in prefrontal ACh levels in mice.

Experimental approach

Apomorphine (1 mg kg⁻¹) was administered to male ddY mice (9–10 weeks old) to create a PPI deficit model. Extracellular ACh concentrations in the prefrontal cortex were measured by in vivo microdialysis.

Key results

Galantamine- and donepezil-mediated improvements in apomorphine-induced PPI deficits were blocked by the preferential M₁ mAChR antagonist telenzepine. The mAChR agonist oxotremorine also improved apomorphine-induced PPI deficits. Galantamine, like donepezil, increased extracellular ACh concentrations in the prefrontal cortex. Galantamine-induced increases in prefrontal ACh levels were partially blocked by the dopamine D₁ receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, but not by antagonists of mAChRs (telenzepine) and nAChRs (mecamylamine). Galantamine increased dopamine, but not 5-HT, release in the prefrontal cortex.

Conclusions and implications

Galantamine improves apomorphine-induced PPI deficits by stimulating mAChRs through increasing brain ACh levels via a dopamine D₁ receptor-dependent mechanism and AChE inhibition.     

Different effects of ascorbate deprivation and classical vascular nitrate tolerance on aldehyde dehydrogenase-catalysed bioactivation of nitroglycerin

Background and purpose:

Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance.

Experimental approach:

Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg·kg⁻¹ GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2-and 1,3-glyceryl dinitrate (GDN).

Key results:

Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN ∼10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the ∼10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance.

Conclusions and implications:

These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.     

Oxidative species increase arginase activity in endothelial cells through the RhoA/Rho kinase pathway

BACKGROUND AND PURPOSE

NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available l-arginine, reducing NO and increasing superoxide (O₂^.−) production via NOS uncoupling. Elevated O₂^.− combines with NO to form peroxynitrite (ONOO^-), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide-induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase (ROCK) pathway.

EXPERIMENTAL APPROACH

Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO^- generator (SIN-1) or H₂O₂. Pretreatment with inhibitors of Rho kinase (Y-27632) or PKC (Gö6976) was used to investigate the mechanism involved in arginase activation.

KEY RESULTS

Exposure to SIN-1 (25 µM, 24 h) or H₂O₂ (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Gö6976 or siRNA to p115-Rho GEF. There was an early activation of p115-Rho GEF (SIN-1, 2 h; H₂O₂, 1 h) and Rho A (SIN-1, 4 h; H₂O₂, 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN-1 and H₂O₂ also reduced NOS activity, which was blocked by pretreatment with p115-RhoGEF siRNA.

CONCLUSIONS AND IMPLICATIONS

Our data indicate that the oxidative species ONOO^- and H₂O₂ increase arginase activity/expression through PKC-mediated activation of RhoA/Rho kinase pathway.     

Pharmacokinetics and dopamine/acetylcholine releasing effects of ginsenoside Re in hippocampus and mPFC of freely moving rats

Aim:

To investigate the pharmacokinetics and dopamine/acetylcholine-releasing effects of ginsenoside Re (Re) in brain regions related to learning and memory, and to clarify the neurochemical mechanisms underlying its anti-dementia activity.

Methods:

Microdialysis was conducted on awake, freely moving adult male SD rats with dialysis probes implanted into the hippocampus, medial prefrontal cortex (mPFC) or the third ventricle. The concentrations of Re, dopamine (DA) and acetylcholine (ACh) in dialysates were determined using LC-MS/MS.

Results:

Subcutaneous administration of a single dose of Re (12.5, 25 or 50 mg/kg) rapidly distributed to the cerebrospinal fluid and exhibited linear pharmacokinetics. The peak concentration (C_max) occurred at 60 min for all doses. Re was not detectable after 240 min in the dialysates for the low dose of 12.5 mg/kg. At the same time, Re dose-dependently increased extracellular levels of DA and ACh in the hippocampus and mPFC, and more prominent effects were observed in the hippocampus.

Conclusion:

The combined study of the pharmacokinetics and pharmacodynamics of Re demonstrate that increase of extracellular levels of DA and ACh, particularly in the hippocampus, may contribute, at least in part, to the anti-dementia activity of Re.     

Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type nicotinic acetylcholine receptor in vitro

Aim:

To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.

Methods:

Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.

Results:

Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC₅₀=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC₅₀=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.

Conclusion:

Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.     

FM19G11 reverses endothelial dysfunction in rat and human arteries through stimulation of the PI3K/Akt/eNOS pathway,independently of mTOR/HIF-1α activation

Background and Purpose

FM19G11 up-regulates mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α) and PI3K/Akt pathways, which are involved in endothelial function. We evaluated the effects of FM19G11 on defective endothelial vasodilatation in arteries from rats and humans and investigated the mechanisms involved.

Experimental Approach

Effects of chronic in vivo administration of FM19G11 on aortic endothelial vasodilatation were evaluated together with ex vivo treatment in aortic and mesenteric arteries from control and insulin-resistant rats (IRR). Its effects on vasodilator responses of penile arteries (HPRAs) and corpus cavernosum (HCC) from men with vasculogenic erectile dysfunction (ED) (model of human endothelial dysfunction) were also evaluated. Vascular expression of phosphorylated-endothelial NOS (p-eNOS), phosphorylated-Akt (p-Akt) and HIF-1α was determined by immunodetection and cGMP by elisa.

Key Results

Chronic administration of FM19G11 reversed the impaired endothelial vasodilatation in IRR. Ex vivo treatment with FM19G11 also significantly improved endothelium-dependent vasodilatation in aorta and mesenteric arteries from IRR. These effects were accompanied by the restoration of p-eNOS and cGMP levels in IRR aorta and were prevented by either NOS or PI3K inhibition. p-Akt and p-eNOS contents were increased by FM19G11 in aortic endothelium of IRR. FM19G11-induced restoration of endothelial vasodilatation was unaffected by mTOR/HIF-1α inhibitors. FM19G11 also restored endothelial vasodilatation in HPRA and HCC from ED patients.

Conclusions and Implications

Stimulation of the PI3K/Akt/eNOS pathway by FM19G11 alleviates impaired NO-mediated endothelial vasodilatation in rat and human arteries independently of mTOR/HIF-1α activation. This pharmacological strategy could be beneficial for managing pathological conditions associated with endothelial dysfunction, such as ED.     

On the selectivity of neuronal NOS inhibitors

Background and Purpose

Isoform-selective inhibitors of NOS enzymes are desirable as research tools and for potential therapeutic purposes. Vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine (l-VNIO) and N^ω-propyl-l-arginine (NPA) purportedly have good selectivity for neuronal over endothelial NOS under cell-free conditions, as does N-[(3-aminomethyl)benzyl]acetamidine (1400W), which is primarily an inducible NOS inhibitor. Although used in numerous investigations in vitro and in vivo, there have been surprisingly few tests of the potency and selectivity of these compounds in cells. This study addresses this deficiency and evaluates the activity of new and potentially better pyrrolidine-based compounds.

Experimental Approach

The inhibitors were evaluated by measuring their effect on NMDA-evoked cGMP accumulation in rodent hippocampal slices, a response dependent on neuronal NOS, and ACh-evoked cGMP synthesis in aortic rings of the same animals, an endothelial NOS-dependent phenomenon.

Key Results

l-VNIO, NPA and 1400W inhibited responses in both tissues but all showed less than fivefold higher potency in the hippocampus than in the aorta, implying useless selectivity for neuronal over endothelial NOS at the tissue level. In addition, the inhibitors had a 25-fold lower potency in the hippocampus than reported previously, the IC₅₀ values being approximately 1 μM for l-VNIO and NPA, and 150 μM for 1400W. Pyrrolidine-based inhibitors were similarly weak and nonselective.

Conclusion and Implications

The results suggest that l-VNIO, NPA and 1400W, as well as the newer pyrrolidine-type inhibitors, cannot be used as neuronal NOS inhibitors in cells without stringent verification. The identification of inhibitors with useable selectivity in cells and tissues remains an important goal.     

Contribution of the M3 muscarinic receptors to the vasodilator response to acetylcholine in the human forearm vascular bed

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Available evidence suggests that the M₃ receptor on endothelial cells is responsible for acetylcholine (Ach)-dependent vasodilatation.
• Data from human studies only provide indirect evidence for this, and results are more difficult to interpret.

WHAT THIS STUDY ADDS

• This study used the new M₃ receptor antagonist darifenacin as a pharmacological ‘tool’ to investigate the role of M₃ receptor in the human forearm circulation.
• It provides evidence for a major role for the M₃ receptors in ACh-dependent vasodilatation in the forearm vascular bed.

AIMS

Acetylcholine (ACh) is a muscarinic agonist that causes receptor-mediated, endothelium-dependent vasodilatation in the forearm vasculature. Previous indirect evidence suggests this effect may be mediated by muscarinic M₃ receptors. Darifenacin is a recently developed antimuscarinic drug with greater M₃ selectivity, and our main objective was to investigate whether darifenacin affects dose-dependent vasodilatation to ACh in the forearm circulation.

METHODS

Healthy subjects were enrolled in two studies designed to assess the effects of atropine and darifenacin on the forearm blood flow (FBF) response to ACh.

RESULTS

In both studies ACh caused similar dose-dependent vasoditation in the forearm vasculature. In study I (5 subjects), the FBF response to ACh was largely attenuated by pretreatment with the nonselective muscarinic antagonist atropine. In study II (10 subjects), oral administration of darifenacin 15 mg for 1 week significantly reduced the FBF dose-dependent response to ACh 20 µg min⁻¹ {mean difference from placebo 5.8 [95% confidence interval (CI) 3.1, 8.7] ml min⁻¹ per 100 ml of forearm volume, P < 0.001} and to ACh 60 µg min⁻¹[mean difference from placebo 5.9 (95% CI 3.1, 8.7) ml min⁻¹ per 100 ml of forearm volume, P < 0.001]. After darifenacin, the AUC of change in FBF from baseline was reduced by almost 50% compared with placebo.

CONCLUSIONS

These results suggest that, in the forearm vasculature, muscarinic M₃ receptors play a major role in ACh-induced endothelium-mediated vasodilatation.     

Sesamin ameliorates arterial dysfunction in spontaneously hypertensive rats via downregulation of NADPH oxidase subunits and upregulation of eNOS expression

Aim:

Sesamin is one of the major lignans in sesame seeds with antihyperlipidemic, antioxidative and antihypertensive activities. The aim of this study was to examine the effects of sesamin on arterial function in spontaneously hypertensive rats (SHRs).

Methods:

SHRs were orally administered sesamin (40, 80 and 160 mg·kg⁻¹·d⁻¹) for 16 weeks. After the rats were killed, thoracic aortas were dissected out. The vasorelaxation responses of aortic rings to ACh and nitroprusside were measured. The expression of eNOS and NADPH oxidase subunits p47^phox and p22^phox in aortas were detected using Western blotting and immunohistochemistry. Aortic nitrotyrosine was measured with ELISA. The total antioxidant capacity (T-AOC) and MDA levels in aortas were also determined.

Results:

The aortic rings of SHRs showed significantly smaller ACh-induced and nitroprusside-induced relaxation than those of control rats. Treatment of SHRs with sesamin increased both the endothelium-dependent and endothelium-independent relaxation of aortic rings in a dose-dependent manner. In aortas of SHRs, the level of T-AOC and the expression of nitrotyrosine, p22^phox and p47^phox proteins were markedly increased, while the level of MDA and the expression of eNOS protein were significantly decreased. Treatment of SHRs with sesamin dose-dependently reversed these biochemical and molecular abnormalities in aortas.

Conclusion:

Long-term treatment with sesamin improves arterial function in SHR through the upregulation of eNOS expression and downregulation of p22^phox and p47^phox expression.     

Reduction in extracellular Ca^2＋ attenuates endothelium-dependent relaxation more than nitroprussideinduced relaxation

Aim:

To quantitatively assess the effect of lowering external Ca²⁺ ([Ca²⁺]_o) on both endothelium-dependent and -independent relaxations in rabbit aorta.

Methods:

Isometric contractions and relaxations of isolated aortae were recorded. When assessing the effect of reduced [Ca²⁺]_o on relaxations, the normal [Ca²⁺]_o solution was substituted with one of the reduced [Ca²⁺]_o solutions for one aorta, while a paired aorta was replenished with normal [Ca²⁺]_o solution.

Results:

The extent of acetylcholine (ACh)-induced relaxation, which is dependent on an intact endothelium, is time-dependent, and inversely related to [Ca²⁺]_o in a range of 0.02–2 mmol/L. ACh-induced relaxations were not significantly altered by the magnitude of the precontraction induced by PGF_2α. Nitroprusside-induced relaxations, which are independent of the endothelium, are also attenuated by reduced [Ca²⁺]_o. Relaxant responses to ACh were significantly more susceptible to reduced [Ca²⁺]_o than nitroprusside-induced relaxations. A maximally effective relaxing concentration of D600, an L-type Ca channel blocker methoxyverapamil, (10⁻⁵ mol/L) attenuated ACh-induced relaxations, whereas nitroprusside-induced relaxations were unaffected by D600.

Conclusion:

Thus, endothelium-dependent relaxation is more dependent on [Ca²⁺]_o than endothelium-independent relaxation, and it seems likely that [Ca²⁺]_o plays an important role not only in contractile processes, but also in relaxant processes as well.     

Role of aldehyde dehydrogenase in hypoxic vasodilator effects of nitrite in rats and humans

Background and Purpose

Hypoxic conditions favour the reduction of nitrite to nitric oxide (NO) to elicit vasodilatation, but the mechanism(s) responsible for bioconversion remains ill defined. In the present study, we assess the role of aldehyde dehydrogenase 2 (ALDH2) in nitrite bioactivation under normoxia and hypoxia in the rat and human vasculature.

Experimental Approach

The role of ALDH2 in vascular responses to nitrite was studied using rat thoracic aorta and gluteal subcutaneous fat resistance vessels from patients with heart failure (HF; 16 patients) in vitro and by measurement of changes in forearm blood flow (FBF) during intra-arterial nitrite infusion (21 patients) in vivo. Specifically, we investigated the effects of (i) ALDH2 inhibition by cyanamide or propionaldehyde and the (ii) tolerance-independent inactivation of ALDH2 by glyceryl trinitrate (GTN) on the vasodilator activity of nitrite. In each setting, nitrite effects were measured via evaluation of the concentration–response relationship under normoxic and hypoxic conditions in the absence or presence of ALDH2 inhibitors.

Key Results

Both in rat aorta and human resistance vessels, dilatation to nitrite was diminished following ALDH2 inhibition, in particular under hypoxia. In humans there was a non-significant trend towards attenuation of nitrite-mediated increases in FBF.

Conclusions and Implications

In human and rat vascular tissue in vitro, hypoxic nitrite-mediated vasodilatation involves ALDH2. In patients with HF in vivo, the role of this enzyme in nitrite bioactivation is at the most, modest, suggesting the involvement of other more important mechanisms.     

Impact of species variability and 'probe-dependence' on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor

BACKGROUND AND PURPOSE

We recently characterized LY2033298 as a novel allosteric modulator and agonist at M₄ muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M₄ mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions.

EXPERIMENTAL APPROACH

LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M₄ mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [³⁵S]-GTPγS binding, or effects on equilibrium binding of [³H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline.

KEY RESULTS

LY2033298 had similar affinities for the human and mouse M₄ mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M₄ mAChR. At the mouse M₄ mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M₄ mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M₄ mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to ‘probe dependence’. This has implications for the validation of allosteric modulator actions in vivo.