l-Tetrahydropalmatine, an active component of Corydalis yanhusuo W.T. Wang, protects against myocardial ischaemia-reperfusion injury in rats

l-Tetrahydropalmatine (l-THP) is an active ingredients of Corydalis yanhusuo W.T. Wang, which protects against acute global cerebral ischaemia-reperfusion injury. In this study, we show that l-THP is cardioprotective in myocardial ischaemia-reperfusion injury and examined the mechanism. Rats were treated with l-THP (0, 10, 20, 40 mg/kg b.w.) for 20 min before occlusion of the left anterior descending coronary artery and subjected to myocardial ischaemia-reperfusion (30 min/6 h). Compared with vehicle-treated animals, the infarct area/risk area (IA/RA) of l-THP (20, 40 mg/kg b.w.) treated rats was reduced, whilst l-THP (10 mg/kg b.w.) had no significant effect. Cardiac function was improved in l-THP-treated rats whilst plasma creatine kinase activity declined. Following treatment with l-THP (20 mg/kg b.w.), subunit of phosphatidylinositol 3-kinase p85, serine(473) phosphorylation of Akt and serine(1177) phosphorylation of endothelial NO synthase (eNOS) increased in myocardium, whilst expression of inducible NO synthase (iNOS) decreased. However, the expression of HIF-1α and VEGF were increased in I(30 min)R(6 h), but decreased to normal level in I(30 min)R(24 h), while treatment with l-THP (20 mg/kg b.w.) enhanced the levels of these two genes in I(30 min)R(24 h). Production of NO in myocardium and plasma, activity of myeloperoxidase (MPO) in plasma and the expression of tumour necrosis factor-α (TNF-α) in myocardium were decreased by l-THP. TUNEL assay revealed that l-THP (20 mg/kg b.w.) reduced apoptosis in myocardium. Thus, we show that l-THP activates the PI3K/Akt/eNOS/NO pathway and increases expression of HIF-1α and VEGF, whilst depressing iNOS-derived NO production in myocardium. This effect may decrease the accumulation of inflammatory factors, including TNF-α and MPO, and lessen the extent of apoptosis, therefore contributing to the cardioprotective effects of l-THP in myocardial ischaemia-reperfusion injury.     

Nitric oxide modulates sympathoexcitatory cardiac-cardiovascular reflexes elicited by bradykinin

A number of studies have demonstrated an important role for nitric oxide (NO) in central and peripheral neural modulation of sympathetic activity. To assess the interaction and integrative effects of NO release and sympathetic reflex actions, we investigated the influence of inhibition of NO on cardiac-cardiovascular reflexes. In anesthetized, sinoaortic-denervated and vagotomized cats, transient reflex increases in arterial blood pressure (BP) were induced by application of bradykinin (BK, 0.1-10 microg/ml) to the epicardial surface of the heart. The nonspecific NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg/kg iv) was then administered and stimulation was repeated. L-NMMA increased baseline mean arterial pressure (MAP) from 129 +/- 8 to 152 +/- 9 mmHg and enhanced the change in MAP in response to BK from 32 +/- 3 to 39 +/- 5 mmHg (n = 9, P < 0.05). Pulse pressure was significantly enhanced during the reflex response from 6 +/- 4 to 27 +/- 6 mmHg after L-NMMA injection due to relatively greater potentiation of the rise in systolic BP. Both the increase in baseline BP and the enhanced pressor reflex were reversed by L-arginine (30 mg/kg iv). Because L-NMMA can inhibit both brain and endothelial NOS, the effects of 7-nitroindazole (7-NI, 25 mg/kg ip), a selective brain NOS inhibitor, on the BK-induced cardiac-cardiovascular pressor reflex also were examined. In contrast to L-NMMA, we observed significant reduction of the pressor response to BK from 37 +/- 5 to 18 +/- 3 mmHg 30 min after the administration of 7-NI (n = 9, P < 0.05), an effect that was reversed by L-arginine (300 mg/kg iv, n = 7). In a vehicle control group for 7-NI (10 ml of peanut oil ip), the pressor response to BK remained unchanged (n = 6, P > 0.05). In conclusion, neuronal NOS facilitates, whereas endothelial NOS modulates, the excitatory cardiovascular reflex elicited by chemical stimulation of sympathetic cardiac afferents.     

Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

Design, synthesis and biological assessment of novel N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines and 3-substituted 2,6-dioxopiperidines for TNF-α inhibitory activity

Eight novel 2-(2,6-dioxopiperidin-3-yl)phthalimidine EM-12 dithiocarbamates 9 and 10, N-substituted 3-(phthalimidin-2-yl)-2,6-dioxopiperidines 11-14 and 3-substituted 2,6-dioxopiperidines 16 and 18 were synthesized as tumor necrosis factor-α (TNF-α) synthesis inhibitors. Synthesis involved utilization of a novel condensation approach, a one-pot reaction involving addition, iminium rearrangement and elimination, to generate the phthalimidine ring required for the creation of compounds 9-14. Agents were, thereafter, quantitatively assessed for their ability to suppress the synthesis on TNF-α in a lipopolysaccharide (LPS)-challenged mouse macrophage-like cellular screen, utilizing cultured RAW 264.7 cells. Whereas compounds 9, 14 and 16 exhibited potent TNF-α lowering activity, reducing TNF-α by up to 48% at 30 μM, compounds 12, 17 and 18 presented moderate TNF-α inhibitory action. The TNF-α lowering properties of these analogs proved more potent than that of revlimid (3) and thalidomide (1). In particular, N-dithiophthalimidomethyl-3-(phthalimidin-2-yl)-2,6-dioxopiperidine 14 not only possessed the greatest potency of the analogs to reduce TNF-α synthesis, but achieved this with minor cellular toxicity at 30 μM. The pharmacological focus of the presented compounds is towards the development of well-tolerated agents to ameliorate the neuroinflammation, that is, commonly associated with neurodegenerative disorders, epitomized by Alzheimer's disease and Parkinson's disease.     

Nitric oxide regulates retinal vascular tone in humans

The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.     

Anti-inflammatory effects of Houttuynia cordata supercritical extract in carrageenan-air pouch inflammation model

Anti-inflammatory effects of Houttuynia cordata supercritical extract (HSE) were investigated in rat carrageenan-air pouch model. Oral administration of HSE (50-200 mg/kg) suppressed carrageenan-induced exudation and albumin leakage, as well as inflammatory cell infiltration at a high dose (200 mg/kg). Intraperitoneal injection of dexamethasone (2 mg/kg) only decreased exudation and cell infiltration, while indomethacin (2 mg/kg, i.p.) reduced exudate volume and albumin content without influence on the cell number. HSE lowered tumor-necrosis factor-α (TNF-α) and nitric oxide (NO), as well as prostaglandin E(2) (PGE(2)). Dexamethasone only reduced TNF-α and NO, while indomethacin decreased PGE(2). The results indicate that HSE exhibits anti-inflammatory effects by inhibiting both TNF-α-NO and cyclooxygenase-2-PGE(2) pathways.     

Nitric oxide and superoxide radical are involved in both initiation and development of adjuvant arthritis in rats

Nitric oxide synthase(NOS) inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 10–300 mg/kg) and L-N^G-monomethyl-arginine (L-NMMA, 30–300 mg/kg) suppressed the swellings of adjuvant-injected paw of rats (25–54%) at day 2 and 8 when dosed intraperitoneally and orally for 4 days from day -1 to day 2 after adjuvant. L-NAME (30–300 mg/kg) also suppressed the edema of the non adjuvant-injected paws (15–42%) at day 28. Local injection of this inhibitor (2 and 10 mg/kg) was without effect. L-arginine (1 g/kg, i.p.), impaired the suppression by L-NAME. Bovine blood Cu, Zn-superoxide dismutase (SOD, 3 mg/kg, i.p.: 28% suppression) and L-NAME (30 mg/kg i.p.: 36% suppression) showed additive effect (52%) in adjuvant-injected paws at day 8 when co-injected. As the effect of 30 mg/kg L-NAME corresponded nearly to that of 10 mg/kg Voltaren^R, this NOS inhibitor would be worth considering as an anti-inflammatory agent. Sodium nitroprusside (NO-donor) and methylene blue (guanylate cyclase inhibitor) had no effect. L-NAME was also suppressive when dosed after adjuvant inoculation and NO is involved in the development and maintenance of swelling.     

Hemodynamic and sympathoadrenal responses to mental stress during nitric oxide synthesis inhibition

Cardiovascular and sympathoadrenal responses to a reproducible mental stress test were investigated in eight healthy young men before and during intravenous infusion of the nitric oxide (NO) synthesis inhibitor N-monomethyl-L-arginine (L-NMMA). Before L-NMMA, stress responses included significant increases in heart rate, mean arterial pressure, and cardiac output (CO) and decreases in systemic and forearm vascular resistance. Arterial plasma norepinephrine (NE) increased. At rest after 30 min of infusion of L-NMMA (0.3 mg.kg(-1).min(-1) iv), mean arterial pressure increased from 98 +/- 4 to 108 +/- 3 mmHg (P <0.001) because of an increase in systemic vascular resistance from 12.9 +/- 0.5 to 18.5 +/- 0.9 units (P <0.001). CO decreased from 7.7 +/- 0.4 to 5.9 +/- 0.3 l/min (P <0.01). Arterial plasma NE decreased from 2.08 +/- 0.16 to 1.47 +/- 0.14 nmol/l. Repeated mental stress during continued infusion of L-NMMA (0.15 mg.kg(-1).min(-1)) induced qualitatively similar cardiovascular responses, but there was a marked attenuation of the increase in mean arterial blood pressure, resulting in similar "steady-state" blood pressures during mental stress without and with NO blockade. Increases in heart rate and CO were attenuated, but stress-induced decreases in systemic and forearm vascular resistance were essentially unchanged. Arterial plasma NE increased less than during the first stress test. Thus the increased arterial tone at rest during L-NMMA infusion is compensated for by attenuated increases in blood pressure during mental stress, mainly through a markedly attenuated CO response and suppressed sympathetic nerve activity.     

Activation of Class I histone deacetylases contributes to mitochondrial dysfunction in cardiomyocytes with altered complex activities

Histone deacetylases (HDACs) play vital roles in the pathophysiology of heart failure, which is associated with mitochondrial dysfunction. Tumor necrosis factor-α (TNF-α) contributes to the genesis of heart failure and impairs mitochondria. This study evaluated the role of HDACs in TNF-α-induced mitochondrial dysfunction and investigated their therapeutic potential and underlying mechanisms. We measured mitochondrial oxygen consumption rate (OCR) and ATP production using Seahorse XF24 extracellular flux analyzer and bioluminescent assay in control and TNF-α (10 ng/ml, 24 h)-treated HL-1 cells with or without HDAC inhibition. TNF-α increased Class I and II (but not Class IIa) HDAC activities (assessed by Luminescent) with enhanced expressions of Class I (HDAC1, HDAC2, HDAC3, and HDAC8) but not Class IIb HDAC (HDAC6 and HDAC10) proteins in HL-1 cells. TNF-α induced mitochondrial dysfunction with impaired basal, ATP-linked, and maximal respiration, decreased cellular ATP synthesis, and increased mitochondrial superoxide production (measured by MitoSOX red fluorescence), which were rescued by inhibiting HDACs with MPT0E014 (1 μM, a Class I and IIb inhibitor), or MS-275 (1 μM, a Class I inhibitor). MPT0E014 reduced TNF-α-decreased complex I and II enzyme (but not III or IV) activities (by enzyme activity microplate assays). Our results suggest that Class I HDAC actions contribute to TNF-α-induced mitochondrial dysfunction in cardiomyocytes with altered complex I and II enzyme regulation. HDAC inhibition improves dysfunctional mitochondrial bioenergetics with attenuation of TNF-α-induced oxidative stress, suggesting the therapeutic potential of HDAC inhibition in cardiac dysfunction.     

Inhibition of LPS-induced TNF-α production by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages

The purpose of this study was to examine whether rCGRP has effects on TNF-α produced by mouse resident peritoneal macrophages. Macrophages were obtained from the peritoneal exudate of male Balb/c mouse. The cells were plated on culture dishes at a density of 2.5 × 10⁵ cells per well and allowed to adhere for 2 hr. Pretreatment with rCGRP (10 nM − 1 μM) for 24 hr, the macrophages were cultured with LPS 1 μg/ml for another 24 h. The medium was harvested for measuring TNF-α by ELISA kits. The results showed that rCGRP had no direct effects on TNF-α production, but it inhibited LPS-induced TNF-α production in a concentration-dependent manner. When rCGRP was at a concentration of 1 μM, the LPS-induced TNF-α production was inhibited by 39%. The effect of rCGRP was reversed by hCGRP8-37 (10 μM), an antagonist of CGRP₁ receptor. The LPS-induced TNF-α production from macrophages was also inhibited by forskolin 3 μM, an activator of adenylate cyclase. Furthermore, pretreatment with H-89 1 μM or Rp-cAMPS 100 μM, the inhibitors of cAMP-dependent protein kinase, the effect of rCGRP was abolished. These data suggest that the LPS-induced TNF-α production is inhibited by rCGRP via activation of cAMP responses in mouse resident peritoneal macrophages.     

Adenosine linking reduced O2 to arteriolar NO release in intestine is not formed from extracellular ATP

We have previously reported that adenosine formed in response to reduced arteriolar and/or tissue PO(2) preserves endothelial nitric oxide (NO) synthesis during sympathetic vasoconstriction in the rat intestine. To more precisely identify the site and mechanism of adenosine formation under these conditions, we tested the hypothesis that ATP released in response to reduced O(2) levels serves as a source of adenosine. Direct application of ATP to the wall of first-order arterioles elicited dose-dependent dilations of 15-33% above resting diameter that were reduced by 71-80% by the 5'-ectonucleotidase inhibitor alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP, 4.5 x 10(-5) M) and completely abolished by N(G)-monomethyl-L-arginine (L-NMMA, 10(-4) M). Under control conditions, sympathetic nerve stimulation at 3 and 8 Hz induced arteriolar constrictions of 11 +/- 1 and 19 +/- 1 microm, respectively. These responses were enhanced by 58-69% in the presence of L-NMMA or when local PO(2) was maintained at resting levels. However, in the presence of AOPCP, the enhancing effects of L-NMMA and the high O(2) superfusate on sympathetic constriction were preserved. These results suggest that, although exogenously applied ATP can stimulate arteriolar NO release in the intestine largely through its sequential extracellular hydrolysis to adenosine, this process does not contribute to adenosine formation and sustained NO release during sympathetic constriction in this vascular bed.     

Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats

The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague-Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day(-1) arginine; group Gln, supplemented with 300 mg/kg day(-1) glutamine; group AG, supplemented with 150 mg/kg day(-1) arginine and 150 mg/kg day(-1) glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P < 0.05) in the jejunum and ileum induced by LPS challenge. LPS administration resulted in a significant increase in TNF-α, IL-1β, IL-6 and IL-10 mRNA abundance. Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.     

Role of nitric oxide in the regulation of glucose kinetics in response to endotoxin in dogs.

The purpose of the present in vivo study was to determine the role of nitric oxide (NO) in the regulation of glucose metabolism in response to endotoxin by blocking NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). In five dogs, the appearance and disappearance rates of glucose (by infusion of [6,6-(2)H(2)]glucose), plasma glucose concentration, and plasma hormone concentrations were measured on five different occasions: saline infusion, endotoxin alone (E coli, 1.0 microg/kg i.v.), and endotoxin administration plus three different doses of primed, continuous infusion of L-NMMA. Endotoxin increased rate of appearance of glucose from 13.7 +/- 1.6 to 23.6 +/- 3.3 micromol x kg(-1) x min(-1) (P < 0.05), rate of disappearance of glucose from 13.9 +/- 1.1 to 24.8 +/- 3.1 micromol x kg(-1) x min(-1) (P < 0.001), plasma lactate from 0.5 +/- 0.1 to 1.7 +/- 0.1 mmol/l (P < 0.01), and counterregulatory hormone concentrations. L-NMMA did not affect the rise in rate of appearance and disappearance of glucose, plasma lactate, or the counterregulatory hormone response to endoxin. Plasma glucose levels were not affected by endotoxin with or without L-NMMA. In conclusion, in vivo inhibition of NO synthesis by high doses of L-NMMA does not affect glucose metabolism in response to endotoxin, indicating that NO is not a major mediator of glucose metabolism during endotoxemia in dogs.     

Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

Nitric oxide potentiates oligosaccharide-induced artemisinin production in Artemisia annua hairy roots

The purpose of the present study was to characterize the generation of nitric oxide （NO） in Artemisia annua roots induced by an oligosaccharide elicitor （OE） from Fusarium oxysporum mycelium and the potentiation role of NO in the elicitation of artemisinin accumulation. The OE （0.3 mg total sugar/mL） induced a rapid production of NO in cultures, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 8 h of OE treatment. Artemisinin content in 20-day-old hairy roots was increased from 0.7mg/g dry wt to 1.3 mg/g dry wt by using the OE treatment for 4d. In the absence of OE, the NO donor sodium nitroprusside （SNP） at 10, 50 ~1 and 100 ~1 enhanced the growth of hairy roots, but had no effect on artemisinin synthesis, The combination of SNP with OE increased artemisinin content from 1.2 mg/g drywt to 2.2 mg/g dry wt, whereas the maximum production of artemisinin in cultures was 28.5 mg/L, a twofold increase over the OE treatment alone. The effects of SNP on the OE-induced artemisinin were suppressed strongly by the NO scavenger 2-（4- carboxyphenyl）-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide （cPTIO）. The results suggest that NO can strongly potentiate elicitor-induced artemisinin synthesis in A. annua hairy roots.     

Effects of inhibition of nitric oxide synthesis on TNFα serum levels in e. coli endotoxemic rats

We investigated the effects of nitric oxide (NO) synthesis inhibition on mortality rate and TNFa serum levels in rats inoculated with E. Coli endotoxin (30 mg/kg i.V.). Pre-treatment of endotoxemic rats with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis by both the constitutive and the inducible isoforms of the NO synthase, did not change the mortality rate but significantly reduced TNFa serum levels. By contrast, administration of aminoguanidine, a more specific inhibitor of the inducible NO synthase, did not modifiy serum TNFα. These results suggest that, in E. Coli endotoxemic rats, NO synthetized by the constitutive isoform of the NO synthase positively modulates TNFa synthesis.     

Mg(2+)-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO

In vitro extracellular Mg(2+) concentration ([Mg(2+)](0)) produces endothelium-dependent and endothelium-independent relaxations in rat aorta in a concentration-dependent manner. These relaxant effects of Mg(2+) on intact rat aortic rings, but not denuded rat aortic rings, were suppressed by either N(G)-monomethyl-L-arginine (L-NMMA), N(omega)-nitro-L-arginine methyl ester (L-NAME), or methylene blue. The inhibitory effects of L-NMMA and L-NAME could be reversed partly by L-arginine. [Mg(2+)](0)-induced dilatation in vivo in rat mesenteric arterioles and venules was almost completely inhibited by N(G)-nitro-L-arginine and L-NMMA. Removal of extracellular Ca(2+) concentration ([Ca(2+)](0)) or buffering intracellular Ca(2+) concentration in endothelial cells, with 10 microM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, markedly attenuated the relaxant effects of Mg(2+). Mg(2+) produced nitric oxide (NO) release from the intact aortic rings in a concentration-dependent manner. Removal of [Ca(2+)](0) diminished the increased NO release induced by elevated levels of [Mg(2+)](0). In vivo infusion of increasing doses (1-30 microM/min) of MgSO(4), directly into the femoral veins of anesthetized rats, elicited significant concentration-dependent sustained increases in serum total Mg and concomitant decreases in arterial blood pressure. Before and after employment of various doses of MgSO(4), intravenous administration of either L-NMMA (10 mg/kg) or L-NAME (10 mg/kg) increased (i.e., reversed) the MgSO(4)-lowered blood pressure markedly, and intravenous injection of L-arginine restored partially the increased blood pressure effects of both L-NMMA and L-NAME. Our results suggest that 1) small blood vessels are very dependent on NO release for Mg(2+) dilatations and 2) the endothelium-dependent relaxation induced by extracellular Mg(2+) is mediated by release of endothelium-derived relaxing factor-NO from the endothelium, and requires Ca(2+) and formation of guanosine 3',5'-cyclic monophosphate.     

Hematopoietic Recovery and Amelioration of Radiation-Induced Lethality by the Vitamin E Isoform δ-Tocotrienol

δ-Tocotrienol (DT3), a vitamin E isoform, is associated with strong antioxidant and immunomodulatory properties. We confirmed the potent antioxidant activity in membrane systems and showed that DT3 is an effective radiation protector and mitigator. DT3 (4 μM, P < 0.001) inhibited lipid peroxidation in mouse liver microsomes and nitric oxide (NO) formation (20 μM DT3, P < 0.01) in RAW264.7 cells, a murine alveolar macrophage line. In CD2F1 mice exposed to lethal total-body radiation from a (60)Co γ-radiation source, a single subcutaneous (s.c.) injection of DT3 before or after irradiation produced a significant increase in 30-day survival. DT3 was effective from 18.75 to 300 mg/kg (--24 h, P < 0.001). A single dose of 150 or 300 mg/kg DT3 given 24 h before irradiation (radioprotection) resulted in dose reduction factors (DRFs) of 1.19 and 1.27, respectively (P < 0.001). Further, DT3 reduced radiation lethality when administered 2, 6 or 12 h after irradiation, and 150 mg/kg DT3 administered 2 h after exposure conferred a DRF of 1.1 (mitigation). The optimum schedule of 300 mg/kg DT3 24 h prior to 7 Gy significantly reduced pancytopenia compared to irradiated controls (P < 0.05). The large therapeutic potential of and multi-lineage hematopoietic recovery for DT3 warrants further studies.     

Rapamycin sensitive ROS formation and Na(+)/H(+) exchanger activity in dendritic cells

Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 μM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 μM) and partially reversed by additional application of tert-butylhydroperoxide (10 μM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.