Anti‐atherogenic effects of high‐density lipoprotein on nitric oxide synthesis in the endothelium

1. The endothelium is critical in the control of vascular haemodynamics and haemostasis. Endothelial dysfunction, typically characterized by decreased nitric oxide bioavailability and response to endothelium‐dependent agonists, is well accepted as a defining characteristic of early atherosclerosis. 2. Numerous epidemiological studies have reported that increased levels of circulating HDL are vasculoprotective and reduce the incidence of adverse cardiovascular events. Traditionally, these effects have been attributed to the ability of HDL to remove cholesterol from cells via reverse cholesterol transport. However, there is increasing evidence that the beneficial effects on the endothelium by HDL encompass its anti‐inflammatory, antithrombotic and anti‐oxidative properties, which include the release of nitric oxide (NO). 3. This review highlights recent findings on the importance of HDL in reducing atherosclerotic risk. We focus on the beneficial effects of HDL‐induced NO release and how this relates to endothelial dysfunction and on the effect of HDL on vascular repair via endothelial progenitor cells.     

Acetaminophen increases the risk of arsenic‐mediated development of hepatic damage in rats by enhancing redox‐signaling mechanism

We evaluated whether the commonly used analgesic‐antipyretic drug acetaminophen can modify the arsenic‐induced hepatic oxidative stress and also whether withdrawal of acetaminophen administration during the course of long‐term arsenic exposure can increase susceptibility of liver to arsenic toxicity. Acetaminophen was co‐administered orally to rats for 3 days following 28 days of arsenic pre‐exposure (Phase‐I) and thereafter, acetaminophen was withdrawn, but arsenic exposure was continued for another 28 days (Phase‐II). Arsenic increased lipid peroxidation and reactive oxygen species (ROS) generation, depleted glutathione (GSH), and decreased superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) activities. Acetaminophen caused exacerbation of arsenic‐mediated lipid peroxidation and ROS generation and further enhancement of serum alanine aminotransferase and aspartate aminotransferase activities. In Phase‐I, acetaminophen caused further GSH depletion and reduction in SOD, catalase, GPx and GR activities, but in Phase‐II, only GPx and GR activities were more affected. Arsenic did not alter basal and inducible nitric oxide synthase (iNOS)‐mediated NO production, but decreased constitutive NOS (cNOS)‐mediated NO release. Arsenic reduced expression of endothelial NOS (eNOS) and iNOS genes. Acetaminophen up‐regulated eNOS and iNOS expression and NO production in Phase‐I, but reversed these effects in Phase‐II. Results reveal that acetaminophen increased the risk of arsenic‐mediated hepatic oxidative damage. Withdrawal of acetaminophen administration also increased susceptibility of liver to hepatotoxicity. Both ROS and NO appeared to mediate lipid peroxidation in Phase‐I, whereas only ROS appeared responsible for peroxidative damage in Phase‐II. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 187–198, 2014.     

Synthesis and antimicrobial evaluation of new nitric oxide‐donating fluoroquinolone/oxime hybrids

A new series of nitric oxide‐donating fluoroquinolone/oximes was prepared in this study. The nitric oxide release from the prepared compounds was measured using a modified Griess colorimetric method. The antitubercular evaluation of the synthesized compounds indicated that ketone derivatives 2b and 2e and oximes 3b and 3d exhibited somewhat higher activity than their respective parent fluoroquinolones. Mycobacterial DNA cleavage studies and molecular modeling of Mycobacterium tuberculosis DNA gyrase were pursued to explain the observed bioactivity. More important, antibacterial evaluation showed that oximes 3c–e are highly potent against Klebsiella pneumoniae, with minimum inhibitory concentration (MIC) values of 0.06, 0.08, and 0.034 µM, respectively, whereas ketone 2c and oxime 4c are more active against Staphylococcus aureus than ciprofloxacin (MIC values: 0.7, 0.38, and 1.6 µM, respectively). Notably, the antipseudomonal activities of compounds 2a and 4c were much higher than those of their respective parent fluoroquinolones.     

The furoxan nitric oxide donor,PRG150, evokes dose‐dependent analgesia in a rat model of painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a type of peripheral neuropathic pain that is often intractable. Elevated nitric oxide (NO) from neuronal and non‐neuronal sources in the somatosensory system is implicated in the pathobiology of peripheral neuropathic pain. However, in diabetes, nitrergic nerve degeneration to deplete NO bioactivity appears causal in the pathogenesis of irreversible autonomic neuropathy, another long term complication of diabetes. Hence, this study hypothesized that progressive NO depletion may underpin the pathobiology of PDN and that NO donors may alleviate PDN. Diabetes was induced in rats with intravenous streptozotocin (STZ) at 70 mg/kg and confirmed if blood glucose levels (BGLs) on day 10 post‐STZ were ≥15 mmol/L. Analgesic efficacy of subcutaneous (s.c.) bolus doses of the furoxan NO donor, PRG150 was assessed in the STZ‐diabetic rat model of PDN at 10‐, 14‐ and 24‐weeks post‐STZ relative to the sydnominine NO donor, SIN‐1 and its prodrug, molsidomine. PRG150 produced dose‐dependent analgesia in STZ‐diabetic rats whereas SIN‐1 and molsidomine evoked neuro‐excitatory side‐effects, but not analgesia. The 1000‐fold larger doses of PRG150 needed to produce analgesia at 14‐ and 24‐weeks (800 pmol/kg) c.f. 10‐weeks (8 fmol/kg) post‐STZ in rats, suggest that progressive NO depletion is also causal in PDN. Importantly, doses of PRG150 up to 10 000 fold higher than the analgesic dose did not produce hypotension in rats. The 50‐fold greater release of NO by SIN‐1 c.f. PRG150 in vitro, may underpin the neuro‐excitatory rather than analgesic effects of SIN‐1/molsidomine. PRG150 is worthy of further investigation as a potential novel analgesic for PDN.     

ZJM‐289, a novel nitric oxide donor,alleviates the cerebral ischaemic–reperfusion injury in rats

1. Current studies indicate that nitric oxide (NO) plays a dual role as both a protective and pathogenic factor in focal cerebral ischaemia depending on the level, location, source and environment. The present study hypothesized that the NO donor ZJM‐289 could inhibit cerebral ischaemia–reperfusion (I/R) injury and investigated the mechanism of the beneficial events. 2. Adult male rats were randomly divided into four groups: (i) sham operated; (ii) I/R (ischaemia for 90 min and reperfusion for 24 h) treated with vehicle; (iii) I/R treated with 0.1 mmol/kg body weight ZJM‐289; and (iv) I/R treated with 0.2 mmol/kg body weight ZJM‐289. We evaluated the changes in brain infarction, brain‐water content, neurological deficits and histopathology. Western blot analysis was used to study the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the brain after I/R. The levels of NO and cyclic guanosine monophosphate (cGMP) were also determined. 3. ZJM‐289 reduced infarct volume and brain‐water content in ischemic brains and promoted functional recovery. Western blotting showed significant inhibition of nNOS in ZJM‐289 treated rats compared with untreated rats. However, eNOS expression in the ischemic brain was enhanced in the ZJM‐289 groups. The cGMP and NO levels increased in the ZJM‐289 groups after I/R. The study showed that ZJM‐289 could alleviate cerebral injury after I/R through inhibition of nNOS and stimulation of the NO/soluble guanylate cyclase/cGMP pathway. Therefore, a suitable NO donor might be an effective candidate for the treatment of acute stroke by neuroprotection.     

Inhibitory effect of fucoidan on nitric oxide production in lipopolysaccharide‐activated primary microglia

1. Microglial activation plays an important role in the pathogenesis of neurodegenerative diseases by producing various pro‐inflammatory cytokines. Microglia‐derived nitric oxide (NO) is critical for the lipopolysaccharide (LPS)‐induced selective loss of dopaminergic neurons. 2. Fucoidan is a sulphated polysaccharide extracted from brown seaweeds. It has a variety of biological actions, including anticoagulant, antiviral and anti‐inflammatory effects. The aim of the present study was to investigate the effects of fucoidan on LPS‐induced cellular activation in microglia and to evaluate the inhibitory mechanisms involved. 3. To investigate the effects of fucoidan on LPS‐induced cellular activation in microglia, primary microglial cells were preincubated with fucoidan (31.25, 62.5 and 125 μg/mL) for 10 min, followed by stimulation with LPS (0.01 μg/mL). Then, cell shape and NO production were determined 24 h after LPS stimulation, whereas inducible nitric oxide synthase (iNOS) mRNA and protein expression were determined at 6 and 18 h after LPS stimulation, respectively. To evaluate the inhibitory mechanisms involved, mitogen‐activated protein kinase (MAPK) activation was also evaluated. 4. Lipopolysaccharide transformed cells into an amoeboid shape, whereas 62.5 μg/mL fucoidan inhibited this activation. Moreover, 125 μg/mL fucoidan significantly inhibited microglial NO production to 75% of that in LPS‐treated group and also significantly diminished the expression of iNOS mRNA and protein by nearly 50%. Fucoidan (125 μg/mL) also suppressed phosphorylation of p38 and extracellular signal‐regulated kinase (ERK) by approximately 50%, but not that of c‐Jun N‐terminal kinase. 5. The results provide the first evidence that fucoidan has a potent inhibitory effect against LPS‐induced NO production by microglia. The results also suggest that this inhibitory action of fucoidan involves suppression of p38 and ERK phosphorylation.     

Thaliporphine protects ischemic and ischemic‐reperfused rat hearts via an NO‐dependent mechanism

In ischemia or ischemia‐reperfusion (I/R), nitric oxide (NO) can potentially exert several beneficial effects. Thaliporphine, a natural alkaloid with Ca²⁺ channel‐activating and Na⁺/K⁺ channel‐blocking activities, increased NO levels and exerted cardioprotective action in ischemic or I/R rats. The role of NO in the cardioprotective actions of thaliporphine was assessed. The severity of rhythm disturbances and mortality in anesthetized rats with either coronary artery occlusion for 30 min, or 5 min followed by 30‐min reperfusion, were monitored and compared in thaliporphine‐ vs. placebo‐treated groups. Thaliporphine treatment significantly increased NO and decreased lactate dehydrogenase (LDH) levels in the blood during the end period of ischemia or I/R. These changes in NO and LDH levels by thaliporphine were associated with a reduction in the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) during ischemic or I/R period. The mortality of animals was also completely prevented by 1 × 10^–8 moles/kg of thaliporphine. In animals subjected to 4 h of left coronary artery occlusion, 1 × 10^–7 moles/kg of thaliporphine dramatic reduced cardiac infarct zone from 46 ± 6% to 7.1 ± 1.9%. Inhibition of NO synthesis with 3.7 × 10^–6 moles/kg of N^ω ‐nitro‐L‐arginine methyl ester (L‐NAME) abolished the beneficial effects of thaliporphine during 30 min or 4 h myocardial ischemia. However, the antiarrhythmic activity and mortality reduction efficacy of thaliporphine during reperfusion after 5 min of ischemia was only partially antagonized by L‐NAME. These results showed that thaliporphine efficiently exerted the cardioprotections either in acute or prolonged coronary artery occlusion or occlusion‐reperfusion situations. The fact that thaliporphine induced cardioprotective effects were abrogated by L‐NAME indicates that NO is an important mediator for the cardioprotective effects of thaliporphine in acute or prolonged ischemia, whereas antioxidant activities may contribute to the protection of I/R injury. Drug Dev. Res. 52:446–453, 2001. © 2001 Wiley‐Liss, Inc.     

Antinociceptive activity of carvacrol (5‐isopropyl‐2‐methylphenol) in mice

Objectives Carvacrol (5‐isopropyl‐2‐methylphenol) is a monoterpenic phenol which is present in the essential oil of oregano and thyme. We have investigated the behavioural effects of carvacrol in animal models of pain, such as acetic acid‐induced abdominal constriction, formalin and hot‐plate tests in mice. The spontaneous motor activity of animals treated with carvacrol was investigated using open‐field and rotarod tests. Methods Carvacrol was administered orally, at single doses of 50 and 100 mg/kg while indometacin (5 mg/kg), morphine (7.5 mg/kg) and diazepam (2 mg/kg) were used as standard drugs. Naloxone (1 mg/kg) and l ‐arginine (150 mg/kg) were used to elucidate the possible antinociceptive mechanism of carvacrol on acetic acid‐induced abdominal constriction and formalin tests. Key findings The results showed that carvacrol produced significant inhibitions on nociception in the acetic acid‐induced abdominal constriction, formalin and hot‐plate tests. In the open‐field and rotarod tests carvacrol did not significantly impair the motor performance. The effect of the highest dose of carvacrol in mice in the acetic acid‐induced abdominal constriction and formalin tests were not reversed by naloxone or l ‐arginine. Conclusions Based on these results, it has been suggested that carvacrol presents antinociceptive activity that may not act through the opioid system nor through inhibition of the nitric oxide pathway.     

Effects of angiotensin receptor blockers on endothelial nitric oxide release: the role of eNOS variants

AIM

Angiotensin II receptor blockers (ARBs) improve endothelial cell (EC)-dependent vasodilation in patients with hypertension through suppression of angiotensin II type 1 receptors but may have additional and differential effects on endothelial nitric oxide (NO) synthase (eNOS) function. To investigate this question, we tested the effects of various ARBs on NO release in ECs from multiple donors, including those with eNOS genetic variants linked to higher cardiovascular risk.

METHODS

The effects of ARBs (losartan, olmesartan, telmisartan, valsartan), at 1 µm, on NO release were measured with nanosensors in human umbilical vein ECs obtained from 18 donors. NO release was stimulated with calcium ionophore (1 µm) and its maximal concentration was correlated with eNOS variants. The eNOS variants were determined by a single nucleotide polymorphism in the promoter region (T-786C) and in the exon 7 (G894T), linked to changes in NO metabolism.

RESULTS

All of the ARBs caused an increase in NO release as compared with untreated samples (P < 0.01, n = 4–5 in all eNOS variants). However, maximal NO production was differentially influenced by eNOS genotype. Olmesartan increased maximal NO release by 30%, which was significantly greater (P < 0.01, n = 4–5 in all eNOS variants) than increases observed with other ARBs.

CONCLUSIONS

The ARBs differentially enhanced NO release in ECs in a manner influenced by eNOS single nucleotide polymorphisms. These findings provide new insights into the effects of ARBs on EC-dependent vasodilation and eNOS function.     

Induction of GSNO reductase but not NOS in the lungs of mice exposed to glucan‐spiked dust

Both in vivo and in vitro studies have suggested that airborne organic dusts may induce inflammatory responses in the lungs, characterized by typical patterns of cytokine up‐regulation and secretion. Recent work showed that exposure to glucan‐spiked dust might influence nasal and pulmonary function, without an accompanying inflammatory response. However, effects of glucan‐spiked dust exposure on NOS and GSNO reductase (enzymes important to NO signaling) remain less clear. This study aims to determine the effects of simultaneous exposure to glucan‐spiked dust on NO signaling pathway in the airway. Danish Office dust was spiked with 1% (1‐3)‐β‐glucan (curdlan). Mice were exposed to 20 μL PBS (controls), 20 μL 25 μg/20 μL OVA and 20 μL 100 μg/20 μL glucan‐spiked dust, respectively, daily for 12 days. NOS and GSNO reductase activity were measured in lung homogenate. Glutathione concentration and SOD activity in lung tissue were also determined to evaluate changes in oxidative stress. IL‐6 concentration was measured in lungs to quantify the inflammatory response. Results showed that 12 day OVA and glucan‐spiked dust exposure did not significantly influence NOS activity, GSH concentration, SOD activity, or IL‐6 concentration. An insignificant increase in GSNOR activity and expression was observed in 12 day OVA‐exposed mice, whereas glucan‐spiked dust exposure significantly increased GSNOR activity and expression. Our results suggested that repeated glucan‐spiked dust exposure to the airway could activate GSNO reductase but not NOS. Since GSNO reductase plays a pivotal role in NO signaling, these results may have clinical importance. © 2009 Wiley Periodicals, Inc. Environ Toxicol 2011.     

Effects of water deprivation and angiotensin II intracerebroventricular administration on brain nitric oxide synthase activity

Intracranial administration of -arginine causes a reduction of the water intake induced by water deprivation or by intracerebroventricular (i.c.v.) injection of angiotensin II (angiotensin II), through the release of nitric oxide (NO) in the central nervous system. We studied the effects of i.c.v. angiotensin II (120 ng/rat) in association with i.c.v. -arginine (2.5–10 μg/rat) on blood pressure. We also studied the effects of both peripheral and central angiotensin II injection (1.5–6 mg kg⁻¹ i.p. and 30–120 ng rat⁻¹ i.c.v., respectively) on NO synthase activity in the cortex, diencephalon and brainstem, after water deprivation (24 h), conditions producing activation of the renin-angiotensin system. -arginine dose dependently antagonized the increase in blood pressure induced by i.c.v. angiotensin II (P<0.001). Peripheral administration of angiotensin II produced a dose-dependent reduction of NO synthase activity in the brainstem and cortex (P<0.001), but not in the diencephalon. Water deprivation produced similar effects on brain NO synthase activity. Angiotensin II i.c.v. injection caused NO synthase activity reduction in all brain regions studied (P<0.001). Our findings suggest that NO and angiotensin II could play opposite roles in brain regulation of blood pressure and drinking behaviour.     

Erythrocyte nitric oxide synthase as a surrogate marker for mercury‐induced vascular damage: The modulatory effects of naringin

In this study, endothelial nitric oxide synthase activity and nitric oxide (NO) production by human erythrocytes in the presence and absence of mercuric chloride (HgCl₂), L ‐arginine (L ‐ARG), ‐ nitro‐L ‐arginine methyl ester (L ‐NAME), and naringin (NAR) were investigated. In addition, the levels of reduced glutathione (GSH) and related enzymes were estimated in erythrocytes hemolysate. The protein carbonyl content (PCC) and thiobarbituric acid‐reactive substances (TBARS) levels were also determined. The results of this study revealed that the treatment of erythrocytes with either HgCl₂ or L ‐NAME induced a significant decrease in NOS activity and nitrite levels compared with control cells. Furthermore, mercury exposure significantly increased the levels of PCC and TBARS but reduced the GSH level. The activities of glucose‐6‐phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and glutathione‐S‐transferase (GST) were inhibited. The exposure of erythrocytes to HgCl₂ in combination with L ‐ARG, NAR, or both ameliorated the investigated parameters compared with erythrocytes incubated with HgCl₂ alone. These results indicate that mercury exposure decreased both erythrocyte NOS activity and nitrite production, and that these parameters might be indicative of mercury exposure. The data also suggest that concomitant treatment with NAR can restore NO bioavailability through either its metal‐chelating properties or its antioxidant activity. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1314–1322, 2014.     

Asymmetric dimethylarginine does not inhibit arginase activity and is pro‐proliferative in pulmonary endothelial cells

Asymmetric dimethylarginine (ADMA) is an endogenously produced nitric oxide synthase (NOS) inhibitor. l ‐Arginine can be metabolised by NOS and arginase, and arginase is the first step in polyamine production necessary for cellular proliferation. We tested the hypothesis that ADMA would inhibit NOS but not arginase activity and that this pattern of inhibition would result in greater l ‐arginine bioavailability to arginase, thereby increasing viable cell number. Bovine arginase was used in in vitro activity assays with various concentrations of substrate (l ‐arginine, ADMA, N^G‐monomethyl‐l ‐arginine (L‐NMMA) and N^G‐nitro‐l ‐arginine methyl ester (l ‐NAME)). Only l ‐arginine resulted in measurable urea production (K_m = 6.9 ± 0.8 mmol/L; V_max = 6.6 ± 0.3 μmol/mg protein per min). We then incubated bovine arginase with increasing concentrations of ADMA, l ‐NMMA and l ‐NAME in the presence of 1 mmol/L l ‐arginine and found no effect of any of the tested compounds on arginase activity. Using bovine pulmonary arterial endothelial cells (bPAEC) we determined the effects of ADMA on nitric oxide (NO) and urea production and found significantly lower NO production and greater urea production (P < 0.003) with ADMA, without changes in arginase protein levels. In addition, ADMA treatment resulted in an approximately 30% greater number of viable cells after 48 h than in control bPAEC. These results demonstrate that ADMA is neither a substrate nor an inhibitor of arginase activity and that in bPAEC ADMA inhibits NO production and enhances urea production, leading to more viable cells. These results may have pathophysiological implications in disorders associated with higher ADMA levels, such as pulmonary hypertension.     

Downregulation of toll‐like receptor 4 and IL‐6 following irradiation of the rat urinary bladder

The pathophysiology behind radiation cystitis is poorly understood. Here we investigated whether bladder irradiation affects the immune system of the rat urinary bladder. Female rats were sedated and exposed to one single radiation dose of 20 Gy or only sedated (controls) and killed 16 h to 14 days later. Rats were placed in a metabolic cage at 16 h, 3 days, 7 days and 14 days following bladder irradiation. The urinary bladders were harvested and analysed with qPCR, immunohistochemistry and/or Western blot for the expression of interferon (IFN)‐γ, interleukin (IL)‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐10, IL‐13, nitric oxide synthases (eNOS, iNOS and nNOS), tumour necrosis factor (TNF)‐α and toll‐like receptor 4 (TLR4). Urine was collected and analysed for IL‐6 and nitrite (reflecting nitric oxide activity) with ELISA and the Griess reaction, respectively. Irradiation increased bladder frequency and decreased voiding volumes 14 days following bladder irradiation. Bladder irradiation increased the expression of IL‐10 and collagen in the bladder, while TLR4 and IL‐6 expressions were decreased in the urothelium concomitantly with a decrease in mast cells in the submucosa and urine levels of IL‐6 and nitrite. The present findings show that bladder irradiation leads to urodynamic changes in the bladder and may suppress important immunoregulatory pathways in the urinary bladder.     

Syntheses,calcium channel modulation effects,and nitric oxide release studies of O2‐alkyl‐1‐(pyrrolidin‐1‐yl) diazen‐1‐ium‐1,2‐diolate 4‐aryl(heteroaryl)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxylates

A group of racemic 4‐aryl(heteroary)‐1,4‐dihydro‐2,6‐dimethyl‐3‐nitropyridine‐5‐carboxy‐lates possessing a potential nitric oxide donor C‐5 O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester [alkyl=(CH₂)_n, n=1–4] substituent were synthesized using a modified Hantzsch reaction. Compounds having a C‐4 2‐trifluoromethylphenyl ( 16 ), 2‐pyridyl ( 17 ), or benzofurazan‐4‐yl ( 20 ) substituent generally exhibited more potent smooth‐muscle calcium channel antagonist activity (IC₅₀ values in the 0.55 to 38.6 μM range) than related analogs having a C‐4 3‐pyridyl ( 18 ), or 4‐pyridyl ( 19 ) substituent with IC₅₀ values > 29.91 μM, relative to the reference drug nifedipine (IC₅₀=0.0143 μM). The point of attachment of C‐4 isomeric pyridyl substituents was a determinant of antagonist activity where the relative potency profile was 2‐pyridyl > 3‐pyridyl and 4‐pyridyl. Subgroups of compounds 16a–d , 17a–d , and 20a–d having alkyl spacer groups of variable chain length [–CO₂(CH₂)_nO–, n=1–4] exhibited small differences in calcium channel antagonist potency. Replacement of the ester “methyl” moiety of Bay K 8644 by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate group provided the Bay K 8644 group of analogs 16a‐d that retained the desired cardiac positive inotropic effect. The most potent compound in this group, O²‐ethyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)pyridine‐5‐carboxylate ( 16b , EC₅₀=0.096 μM) is about eightfold more potent positive inotrope (cardiac calcium channel agonist) than the reference compound Bay K 8644 (EC₅₀=0.77 μM). A similar replacement of the ester “isopropyl” group in the C‐4 benzofurazan‐4‐yl group of compounds by an O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester substituent provided compounds 20 (n=1 and 4) that were approximately equipotent cardiac positive inotropes with the parent reference compound PN 202‐791 ( 3 , EC₅₀=9.40 μM). The O²‐alkyl‐1‐(pyrrolidin‐1‐yl)diazen‐1‐ium‐1,2‐diolate ester moiety present in 1,4‐dihydropyridine calcium channel modulating compounds 16–20 is not a suitable ?NO donor moiety because the percent nitric oxide released upon in vitro incubation with either l ‐cysteine, rat serum, or pig liver esterase was less than 1%. Drug Dev. Res. 60:204–216, 2003. © 2003 Wiley‐Liss, Inc.     

CARDIOVASCULAR PROTECTION BY GINSENOSIDES AND THEIR NITRIC OXIDE RELEASING ACTION

1. In an animal model in vivo, ginsenosides (GS), saponins from Panax ginseng, were shown to protect against myocardial ischaemia/reperfusion damage with concomitant increased 6-keto-PGF_1α and decreased lipid peroxidation. 2. In perfused rabbit lung in situ and isolated rabbit aortic rings, GS protected the pulmonary and aortic endotheluim against electrolysis-induced free radical injury. Purified components of GS, Rb1 and especially Rg1, relaxed pulmonary vessels and this effect was eliminated by nitro-L-arginine, an inhibitor of nitric oxide (NO) synthase. 3. In cultured bovine aortic endothelial cells, GS enhanced the conversion of [¹⁴C]-L-arginine to [¹⁴C]-L-citrulline, indicating an increased release of NO. 4. As the neurotransmitter inducing penile erection, NO release was shown to be enhanced by GS in rabbit corpus cavernosum (CC) in vitro. Ginsenosides enhanced both acetylcholine-induced and transmural nerve stimulation-activated relaxation associated with increased tissue cGMP. The latter effect was eliminated by tetrodotoxin and was associated with decreased tissue cGMP. Ginsenoside-enhanced CC relaxation was attenuated by nitro-L-arginine and oxyhaemoglobin, and enhanced by superoxide dismutase. 5. It is postulated that cardiovascular protection by GS may be partly mediated by the release of NO, a potent antioxidant, and that the GS-enhanced release of NO from endothelial cells, especially from perivascular nitric oxidergic nerves in the CC, may partly account for the aphrodisiac effect of Panax ginseng used in traditional Chinese medicine.     

Effects of nitric oxide on large‐conductance Ca2+‐activated K+ currents in human cardiac fibroblasts through PKA and PKG‐related pathways

The human cardiac fibroblast (HCF) is the most abundant cell type in the myocardium, and HCFs play critical roles in maintaining normal cardiac function. However, unlike cardiomyocytes, the electrophysiology of HCFs is not well established. In the cardiovascular system, Ca²⁺‐activated K⁺ (KCa) channels have distinct physiological and pathological functions, and nitric oxide (NO) plays a key role. In this study, we investigated the potential effects of NO on KCa channels in HCFs. We recorded strong oscillating, well‐maintained outward K⁺ currents without marked inactivation throughout the test pulse period and detected outward rectification in the I‐V curve; these are all characteristics that are typical of KCa currents. These currents were blocked with iberiotoxin (IBTX, a BKCa blocker) but not with TRAM‐34 (an IKCa blocker). The amplitudes of the currents were increased with SNAP (an NO donor), and these increases were inhibited with IBTX. The SNAP‐stimulating effect on the BKCa currents was blocked by pretreatment with KT5823 (a protein kinase G [PKG] inhibitor) or 1 H‐[1,‐2, ‐4] oxadiazolo‐[4,‐3‐a] quinoxalin‐1‐one (ODQ; a soluble guanylate cyclase inhibitor). Additionally, 8‐bromo‐cyclic guanosine 3’,5’‐monophosphate (8‐Br‐cGMP) stimulated the BKCa currents, and pretreatment with KT5720 (a protein kinase A [PKA] inhibitor) and SQ22536 (an adenylyl cyclase inhibitor) blocked the NO‐stimulating effect on the BKCa currents. Furthermore, 8‐bromo‐cyclic adenosine 3’,5’‐monophosphate (8‐Br‐cAMP) activated the BKCa currents. These data suggest that BKCa current is the main subtype of the KCa current in HCFs and that NO enhances these currents through the PKG and PKA pathways.     

Involvement of nitric oxide synthesis in hepatic perturbations induced in rats by a necrogenic dose of thioacetamide

1. The biological actions of nitric oxide (NO), a highly diffusible and short-lived radical, range from signal transduction to cytotoxicity. The present study investigated whether NO is released in the course of liver necrosis and regeneration induced by a single necrogenic dose of thioacetamide (6.6 mmol kg⁻¹ body wt) to rats. Samples of liver were obtained at 0, 3, 12, 24, 48, 72 and 96 h after thioacetamide administration.
2. Inducible nitric oxide synthase (iNOS) activity was determined in purified liver homogenates and a sharp 6 fold increase (P<0.001) in iNOS activity was recorded at 48 h of intoxication, followed by a slight but progressive increase at 72 and 96 h. Changes in the expression of iNOS, as detected by its mRNA levels, were parallel to the NOS enzyme activity. Hepatocyte NO synthesis showed a progressive increase at 24, 48 and 72 h, to 8 (P<0.001), 13 (P<0.001) and 13 (P<0.001) times the initial values, respectively.
3. In isolated Kupffer cells, where initial NO release was ten fold higher than in hepatocytes, a progressive increase was detected from 48 h which reached two fold of initial at 72 h of intoxication (192%, P<0.001). Hepatic cyclic GMP concentration did not change significantly. However, mitochondrial aconitase activity decreased markedly at 12 and 24 h of intoxication showing a sharp increase towards normal values at 48 h which was maintained at 72 and 96 h.
4. As protein kinase C (PKC) is one of the likely candidates to mediate iNOS expression, translocation (activation) of PKC was assayed in hepatocytes, and a significant two fold increase (P<0.001) between 48 and 96 h after thioacetamide intoxication was observed. When peritoneal macrophages from control rats were incubated with serum from thioacetamide-treated rats, a sharp increase in NO release was detected with serum obtained at 48 h, reaching at 96 h a value four fold (P<0.001) that of the control.
5. These results suggest that iNOS activity and NO release play a role in the pathophysiological mechanisms that trigger post-necrotic hepatocellular regeneration following thioacetamide administration.