Nitro L-arginine Research Articles

Interactions between apelin and angiotensin II on rat portal vein

Apelin (AP), an endogenous ligand of the APJ receptor, is involved in the regulation of cardiovascular homeostasis. Regardless the multiple similarities between AP and angiotensin II (Ang II), their roles seem to be different. We studied both the interactions between Apelin 13 (AP13) and Ang II and to what extent, if any, nitric oxide (NO) is involved. The experiments were performed in endothelium-denuded or endothelium-intact rat portal vein in the presence of 10 microM N(G)-nitro L-arginine methyl ester or 10 microM aminoguanidine. AP13 did not modify the isolated rat portal vein tone by itself, but inhibited the Ang II-induced contractions acting mainly by a NO-dependent mechanism. Our results sustain the hypothesis that AP13 could increase the activity of both constitutive and inducible NO synthase on either endothelium intact or endothelium denuded rat portal vein rings.

Ibuprofen or ozagrel increases NO release and l-nitro arginine induces TXA 2 release from cultured porcine basilar arterial endothelial cells

The vascular resting tone of the porcine basilar artery appears to be mostly maintained by a balance between spontaneously released nitric oxide (NO) from endothelial cells and thromboxane (TX) A 2 from endothelial and smooth muscle cells. However the precise role of the interaction between the above two substances in the control of vascular tone is unclear. We attempted to clarify the interaction between NO and TXA 2 using cultured porcine basilar arterial endothelial cells. The cultured endothelial cells produced NO spontaneously, while TXB 2 (a stable metabolite of TXA 2) production remained below the detection limit. Ibuprofen (a COX inhibitor) and ozagrel (a TXA 2 synthetase inhibitor) significantly increased the spontaneous production of NO, which was not affected by 1400W (an iNOS inhibitor). l-Nitro arginine (a NOS inhibitor) significantly induced TXB 2 production. These results suggest that NO may inhibit COX or TXA 2 synthetase, and that therefore inhibition of NOS might disinhibit COX or TXA 2 synthetase, subsequently inducing TXA 2 production. On the other hand, as TXA 2 and other contractility-related prostaglandin(s) may inhibit NOS, therefore the inhibition of COX or TXA 2 synthetase might disinhibit NOS, and then increase the spontaneous production of NO in porcine basilar arterial endothelial cells.

Muscle microvascular oxygenation in chronic heart failure: role of nitric oxide availability

To test the hypothesis that diminished vascular nitric oxide availability might explain the inability of individuals with chronic heart failure (CHF) to maintain the microvascular PO(2)'s (PO(2mv) proportional, variant O(2) delivery-to-uptake ratio) seen in healthy animals. We superfused sodium nitroprusside (SNP; 300 microm), Krebs-Henseleit (control, CON) and L-nitro arginine methyl ester (L-NAME; 1.5 mM) onto the spinotrapezius muscle and measured PO(2mv) by phosphorescence quenching in female Sprague-Dawley rats (n = 26) at rest and during twitch contractions (1 Hz). Seven rats served as controls (Sham) while CHF was induced by myocardial infarction. CHF rats were grouped as moderate (MOD; n = 15) and severe CHF (SEV; n = 4) according to morphological data and baseline PO(2mv). In contrast to Sham and MOD, L-NAME did not affect the PO(2mv) response (dynamics and steady-state) of SEV when compared with CON. SNP restored the PO(2mv) profile of SEV to that seen in Sham animals during CON. Specifically, the effect of L-NAME expressed as Delta(L-NAME - CON) were: Baseline PO(2mv) [in mmHg, DeltaSham = -7.0 +/- 1.6 (P < 0.05); DeltaSEV =-1.2 +/- 2.1], end-contractions PO(2mv) [in mmHg, DeltaSham = -5.0 +/- 1.0 (P < 0.05); DeltaSEV = -2.5 +/- 0.5] and time constant of PO(2mv) decrease [in s, DeltaSham = -6.5 +/- 3.0 (P < 0.05); DeltaSEV = -3.2 +/- 1.8]. These data provide the first direct evidence that the pathological profiles of PO(2mv) associated with severe CHF can be explained, in part, by a diminished vascular NO availability.

Post-prandial insulin sensitization. new perspectives for gastrointestinal endocrinology

Eating a meal has been found to activate a parasympathetic reflex in the liver that through the release of a hepatic insulin sensitizing substance (HISS) into the circulation sensitizes peripheral tissues to the hypoglycaemic effect of insulin. Partial hepatic sensory denervation (PHSD) was achieved by a 3-day perineurial treatment with 2% capsaicin solution around the anterior hepatic plexus in male guinea-pigs, rats or rabbits to confirm the sensory nature of the HISS mechanism in these species. One week later, hyperinsulinaemic (100µU/ml) euglycaemic (5.5 mmol/l) glucose clamp were performed to estimate whole body insulin sensitivity in anaesthetized animals. Calcitonin gene-related peptide (CGRP), substance P, somatostatin and insulin were determined by means of RIA. The HISS effect was characterized by insulin-stimulated uptake of 2-deoxy-D [l-14C] glucose in cardiac and gastrocnemius muscle and percentage suppression of endogenous glucose production. PHSB decreased post-prandial insulin sensitivity characterized by a decrease in glucose infusion rate from 19±2.4 to 7.9±1.5mg/kg/min to maintain euglycaemia at clamped hyperinsulinaemia. Inhibition of hepatic NO synthesis by either intraportal nitro L-arginine (10mg/kg) or 7-nitro indazole (5mg/kg) attained similar inhibition. Both intraportal and systemic atropine (1mg/kg) was without effect in each species. Post-prandial insulin sensitization (65±7.2% vs. the fasted state) was accompanied by a significant increase in plasma somatostatin immunoreactivity with no change with respect to the other peptides measured. Cysteamine (200mg/kg/s.c.) completely blocked post-prandial insulin sensitization. PHSB decreased post-prandial cardiac 2-deoxy-D [l-14C] glucose uptake by 37±4.6% with no significant effect on hepatic glucose production. The results shed light on a novel hepatic sensory nitrergic endogenous insulin sensitizing mechanism that is mediated by a circulating substance showing somatostatin-like immunoreactivity in three species.

Effects of Altered Nitric Oxide Availability on Rat Muscle Microvascular Oxygenation During Contractions

Aging, chronic heart failure (CHF) and diabetes are all associated with vascular endothelial dysfunction. Compared with muscles from young healthy individuals, those from old and CHF/diabetic individuals exhibit a reduced muscle microvascular O pressure (POmv) at the onset of contractions that is expected to impair blood-myocyte O2 exchange. PURPOSE: To explore the putative role of nitric oxide (NO) in controlling PO2mv at rest and during contractions, we tested the hypotheses that at the onset of contractions (1 Hz) sodium nitroprusside (SNP, ↑ NO) would raise PO2mv and slow the kinetics of PO mv change whereas L-nitro arginine methyl ester (L-NAME, ↓ NO) would decrease PO2mv and speed its kinetics. This would assess the potential for derangements in NO production to induce the PO2mv and O2 kinetics impairments characteristic of aged and CHF/diabetic individuals. METHODS: Separately, we superfused the spinotrapezius muscle of female Sprague-Dawley rats (n = 7, body mass = 298 ± 10 g) with a NO donor SNP (300 uM) and the NO synthase inhibitor L-NAME (1.5 μM) during muscle contractions. Microvascular PO2 was measured by phosphorescence quenching. RESULTS: SNP decreased mean arterial pressure (92 ± 5 mmHg) below that of control (CON, 124 ± 4 mmHg) and L-NAME (120 ± 4 mmHg) conditions. SNP did not raise PO mv at rest but it did slow the kinetics of PO2mv by lengthening the time delay (TD, 14 ± 5 s), time constant (τ, 24 ± 10 s) and mean response time (MRT, 38 ± 4 s) of the response compared with CON (TD, 8.4 ± 3.3 s; τ, 16 ±4.5 s and MRT, 25 ± 1.5 s, P <: 0.05). In contrast, L-NAME decreased PO2mv at rest and speeded τ (10.1 ±3.8 s, P= 0.05), while TD (8.1 ± 1 s) was not significantly different. However, the MRT (18 ± 1 s) was faster for L-NAME compared to CON. Moreover, L-NAME caused PO2mv to transiently fall below steady-state values. CONCLUSION: These results indicate that NO availability can significantly affect microvascular O2 pressures, and therefore O2 transport, at rest and during contractions and suggests that such derangements in aging and chronic disease conditions may potentially result from impairments in NO availability.

Fluorescence Monitoring of ATP-Stimulated, Endothelium-Derived Nitric Oxide Production in Channels of a Poly(dimethylsiloxane)-Based Microfluidic Device

Intracellular nitric oxide (NO) production in a microfluidic endothelium is detected using fluorescence microscopy. Bovine pulmonary artery endothelial cells (bPAECs) were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent fluorescent DAF-FM DA/NO adduct was measured. Solutions of bradykinin, a well-known stimulus of endothelium-derived NO, activated nitric oxide synthase (NOS) in the immobilized bPAECs. This activation was inhibited using l-nitro arginine methyl ester (L-NAME), a competitive inhibitor of NOS. Importantly, the NO production was also stimulated with adenosine triphosphate (ATP) using concentrations as low as 1 microM. Previous reports on stimulating NO production using an immobilized endothelium in microfluidic channels were limited by the requirement of ATP concentrations of at least 100 microM, a value that is not physiologically relevant. The ability to monitor NO production with ATP concentrations that are similar to in vivo levels of ATP in the microcirculation represents a major advance in the use of microfluidic technology as an in vitro model of the microcirculation.

Involvement of endogenous nitric oxide in myeloperoxidase mediated benzo(a)pyrene induced polymorphonuclear leukocytes injury

The present study was undertaken to investigate the involvement of nitric oxide in the augmentation of benzo(a)pyrene induced cellular injury in polymorphonuclear leukocytes (PMNs). Polymorphs were isolated from the blood collected from Wistar rats treated with and without benzo(a)pyrene (50mg/kg, i.p.) through cardiac puncture. Catalase, superoxide dismutase (SOD), glutathione-s-transferase (GST), myeloperoxidase (MPO) and nitrite content were estimated in PMNs using standard procedures. Inducible nitric oxide synthase (iNOS) and cytochrome P-4501A1 (CYP1A1) expression in PMNs were also analyzed in presence or absence of nitric oxide synthase (NOS) inhibitors, aminoguanidine (AG, 5mM) and L-NG nitro L-arginine methyl ester (L-NAME, 1mM). A significant augmentation was observed in the nitrite content, activities of superoxide dismutase, MPO and GST and the expressions of iNOS and CYP1A1, however, catalase activity was attenuated in PMNs of benzo(a)pyrene treated rats as compared with their respective controls. AG and L-NAME resulted in a significant attenuation in nitrite content, MPO activity and iNOS expression; however, no significant alteration was observed in CYP1A1 expression. CYP1A1 inhibitor alpha-naphthoflavone inhibited the expression of iNOS in PMNs of benzo(a)pyrene treated animals significantly. The results obtained thus suggest that CYP1A1 induces iNOS expression leading to the generation of endogenous nitric oxide (NO) that could be responsible for the augmentation of myeloperoxidase-mediated benzo(a)pyrene-induced injury in PMNs.

Effects of nitric oxide on skeletal muscle microvascular O 2 pressure of rats with heart failure

Chronic heart failure (CHF) is associated with reduced vascular nitric oxide (NO) availability that could affect muscle microvascular O2 pressure (PO2mv) during contractions. We tested the hypothesis that diminished NO availability has a role in the pathological profile of PO2mv in CHF. Spinotrapezius PO2mv was measured (phosphorescence quenching) in female Sprague-Dawley rats 6–10 wk post-myocardial infarction (MI, n = 21; Sham, n = 7) during sodium nitroprusside (SNP, 300 μM), control (CON) and L-nitro arginine methyl ester (L-NAME, 1.5 mM) superfusion. Moderate (n = 15) and Severe (n = 6) CHF were defined based on structural abnormalities (i.e., lung and right-ventricular weights normalized to body weight). Unlike in healthy rats during CON, an undershoot in the PO2mv response, reflecting a dynamic mismatch between O2 delivery (QO2) and uptake (VO2), was seen in CHF rats during SNP (n = 2), CON (n = 12) and L-NAME (n = 19). The results (mean ± SE) for end-contraction PO2mv are shown below. These data demonstrate that despite down-regulation of endothelial NO synthase in CHF, NO still plays an important role in matching O2 delivery to O2 uptake in severe CHF. Supported by AHA (0455582Z)

Enhanced Superoxide Activity Modulates Renal Function in NO-Deficient Hypertensive Rats

An enhancement of superoxide (O2-) activity was shown to contribute to the development of hypertension induced by NO deficiency. To better understand the mechanistic role of O2- in this NO-deficient hypertension, we evaluated the renal responses to acute intraarterial administration of an O2- scavenger, tempol (50 microg/min per 100 g of body weight) in anesthetized male Sprague-Dawley rats treated with NO synthase inhibitor nitro-L-arginine methyl ester (15 mg/kg per day in drinking water, n=7) for 4 weeks, which caused increases in mean arterial pressure (146+/-3 versus 124+/-2 mm Hg) compared with normotensive control rats (n=6). Hypertensive rats had higher renal vascular resistance (29+/-2 versus 20+/-1 mm Hg/mL per minute per gram), as well as lower renal blood flow (5.2+/-0.3 versus 6.3+/-0.2 mL/min per gram; cortical blood flow, 153+/-13 versus 191+/-8 perfusion units; medullary blood flow, 43+/-2 versus 51+/-3 perfusion units) and glomerular filtration rate (0.69+/-0.04 versus 0.90+/-0.05 mL/min per gram) without a significant difference in urinary sodium excretion (0.81+/-0.07 versus 0.86+/-0.12 micromol/min per gram) compared with normotensive rats. Urinary 8-isoprostane excretion rate (6.8+/-0.7 versus 4.5+/-0.3 pg/min per gram) was higher in hypertensive than normotensive rats. Intraarterial infusion of tempol did not alter renal function in normotensive rats. However, tempol significantly decreased renal vascular resistance by 12+/-2% and urinary 8-isoprostane excretion rate by 24+/-4% and increased renal blood flow by 10+/-2%, cortical blood flow by 9+/-2%, medullary blood flow by 15+/-6%, glomerular filtration rate by 11+/-3%, and urinary sodium excretion by 19+/-5% in hypertensive rats. These data indicate that enhanced O2- activity modulates renal hemodynamics and excretory function during reduced NO production and, thus, contributes to the pathophysiology of the NO-deficient form of hypertension.

L-NAME prevents anxiety-like and depression-like behaviour in rats exposed to restraint stress

Stressful life events contribute to the development of many neuropsychiatric disorders including depression and anxiety. Animal studies based on the relationship of stress and depression or anxiety are scarce and controversial. Moreover, neither the neurobiological basis of anxiety and depression nor the mechanisms responsible for neurochemical regulation by stressful stimuli are well understood. This study was designed to investigate the possible contribution of both acute (2 h) and chronic (2 h X 15 d) restraint stress in the generation of anxiety and depression, and also to find out whether nitric oxide (NO) has a modulatory role in these behavioral reactions. Elevated plus-maze and forced swimming test (FST) were chosen for assessment of anxiety and depression, respectively, and N(G)-nitro L-arginine methyl ester (L-NAME, 10 mg/kg), a NO synthase (NOS) inhibitor, and L-arginine (50 mg/kg), a NO precursor, were used to evaluate the role of nitrergic system in restraint exposed rats. The results showed that acute and chronic stress caused depression-like and anxiety-like behaviors in rats and the acute inhibition of NOS by L-NAME prevented these acute and chronic stress-induced anxiogenesis and depression. These data lead to the conclusion that stress and NO seem to be involved in the generation of anxiety and depression.

Pregnancy Prevents Hypertensive Remodeling of Cerebral Arteries

We investigated how hypertension during pregnancy affected passive structural (wall:lumen, wall stress) and active (myogenic activity) responses of the cerebral circulation. Female nonpregnant (NP; n=8) Sprague Dawley rats were compared with late-pregnant (LP; day 19 to 20, n=6) rats. Some animals were treated with the NO synthase inhibitor nitro-L-arginine in their drinking water to raise blood pressure. LP rats (n=6) were treated for the last 7 days of pregnancy (last trimester) to mimic preeclampsia and compared with NP rats treated for the same duration (n=8). Active and passive responses were determined on isolated and pressurized third-order posterior cerebral arteries. Nitro-L-arginine treatment significantly raised blood pressure in both groups of animals that was associated with increased wall thickness and wall:lumen ratio in the NP hypertensive animals versus controls (P<0.05). In contrast, this response to pressure was absent in LP animals, which had similar wall measurements. In addition, arteries from NP hypertensive animals had increased myogenic tone and pressure of forced dilatation compared with NP control animals (P<0.01). Again, this response was lacking in the LP hypertensive animals that had similar tone and pressure of forced dilatation as normotensive controls. The increased tone and wall thickness decreased wall stress in the NP hypertensive animals, a response that did not occur in LP hypertensive animals. Because medial hypertrophy is considered a protective response to elevated blood pressure, these results suggest that hypertension in pregnancy may predispose the cerebral circulation to autoregulatory breakthrough and blood-brain-barrier disruption when blood pressure is elevated, as during eclampsia.

Possible Role of Nitric Oxide in the Protective Effect of Resveratrol in 5/6th Nephrectomized Rats

Nitric oxide (NO) plays an important role in the modulation of glomerular disease. The renal protective effect of resveratrol (RVT), a polyphenolic phytoalexin, was investigated in the 5/6th nephrectomized rats. Resveratrol (5 mg/kg, PO) was administered for 12 weeks to 5/6th nephrectomized (NX) rats together with and without nitro L-arginine methyl ester (L-NAME) (10 mg/kg, IP). We evaluated the effect of these agents on proteinuria, hypertension, renal function, glomerulosclerosis, and urinary excretion of nitric oxide metabolites. 5/6th NX resulted in elevation in systolic blood pressure (SBP), reduced the urinary excretion of NO metabolites, increased urinary protein excretion, and deranged renal function and glomerulosclerosis. Treatment of animals with resveratrol significantly attenuated the increase in SBP, preserved the normal renal function, reduced the urinary protein excretion, increased the urinary excretion of NO metabolites, and prevented the glomerulosclerosis. Co-administration of animals with L-NAME along with resveratrol prevented the protection observed with resveratrol. These findings indicate that resveratrol exerts its protective effect in 5/6 NX rats through a nitric oxide pathway.

Endogenous Vascular Hydrogen Peroxide Regulates Arteriolar Tension In Vivo

Although many studies suggested direct vasomotor effects of hydrogen peroxide (H2O2) in vitro, little is known about the vasomotor effects of H2O2 in vivo. We have generated mice overexpressing human catalase driven by the Tie-2 promoter to specifically target this transgene to the vascular tissue. Vessels of these mice (cat++) expressed significantly higher levels of catalase mRNA, protein, and activity. The overexpression was selective for vascular tissue, as evidenced by immunohistochemistry in specimens of aorta, heart, lung, and kidney. Quantification of reactive oxygen species by fluorescence signals in cat++ versus catalase-negative (catn) mice showed a strong decrease in aortic endothelium and left ventricular myocardium but not in leukocytes. Awake male cat++ at 3 to 4 months of age had a significantly lower systolic blood pressure (sBP, 102.7+/-2.2 mm Hg, n=10) compared with their transgene-negative littermates (catn, 115.6+/-2.5 mm Hg, P=0.0211) and C57BL/6 mice (118.4+/-3.06 mm Hg, n=6). Treatment with the catalase inhibitor aminotriazole increased sBP of cat++ to 117.3+/-4.3 mm Hg (P=0.0345), while having no effect in catn (118.4+/-2.4 mm Hg, n=4, P>0.05). In contrast, treatment with the NO-synthase inhibitor nitro-L-arginine methyl ester (100 mg.kg BW(-1).d(-1)) increased sBP in cat++ and C57Bl/6 to a similar extent. Likewise, phosphorylation of vasodilator-stimulated phosphoprotein in skeletal muscle, left ventricular myocardium, and lung was identical in cat++ and catn. Endothelium- and NO-dependent aortic vasodilations were unchanged in cat++. Aortic KCl contractions were significantly lower in cat++ and exogenous H2O2 (10 micromol/L)-induced vasoconstriction. These data suggest that endogenous H2O2 may act as a vasoconstrictor in resistance vessels and contribute to the regulation of blood pressure.

Superoxide Contributes to Development of Salt Sensitivity and Hypertension Induced by Nitric Oxide Deficiency

This study was performed to examine the role of superoxide (O2-) in the development of salt sensitivity and hypertension induced by inhibition of nitric oxide (NO) generation. Male Sprague-Dawley rats were fed with diet containing either normal salt (NS) (0.4% NaCl) or high salt (HS) (4% NaCl). These rats were treated with or without an NO synthase inhibitor, nitro-L-arginine methylester (L-NAME) (15 mg/kg/d) and O2- scavenger, tempol (30 mg/kg per day) in the drinking water for 4 weeks. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and urine collection was performed during the course of experimental periods. At the end of 4 weeks, L-NAME treatment resulted in greater increases in SBP in HS rats (127+/-2 to 172+/-3 mm Hg; n=8) than in NS rats (130+/-2 to 156+/-2 mm Hg; n=9). Co-administration of tempol with L-NAME markedly attenuated these SBP responses to a similar level in both HS (128+/-3 to 147+/-2 mm Hg; n=8) and NS rats (126+/-2 to 142+/-3 mm Hg; n=8). Urinary 8-isoprostane excretion (UIsoV) increased in response to L-NAME treatment that was higher in HS (10.6+/-0.5 to 21.5+/-0.8 ng/d) than in NS rats (10.8+/-0.7 to 16.9+/-0.6 ng/d). Co-treatment with tempol completely abolished these UIsoV responses to L-NAME in both HS and NS rats but did not alter urinary H2O2 excretion rate. The decreases in urinary nitrate/nitrite excretion in response to L-NAME treatment were not altered by co-administration of tempol in both HS and NS rats. These data suggest that enhancement of O2- activity during NO inhibition contributes to the development of salt sensitivity that is associated with NO-deficient hypertension.

Gastric HCO3− secretion induced by mucosal acidification: Different mechanisms depending on acid concentration

We compared the HCO3- secretory responses induced by mucosal acidification at different HCl concentrations (100 and 200 mM HCl) in the rat stomach. Under urethane anesthesia, the stomach was mounted on an ex vivo chamber and perfused with saline under inhibition of acid secretion by omeprazole (60 mg/kg, i.p.). TheHCO3- secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The acidification was performed by exposure of the mucosa to 100 mMor 200 mM HCl for 10 min. The secretion of HCO3- was increased by acidification of the mucosa at both 100 and 200 mM of HCl, and the maximal HCO3- response was 1.5-times greater at the latter concentration. The HCO3- responses induced by 100 and 200 mM HCl were both totally inhibited by prior administration of indomethacin, an inhibitor of prostaglandin (PG) production. The HCO3- stimulatory effect of 200 mM HCl was also significantly attenuated by pre-treatment with N(G)-nitro L-arginine methyl ester (L-NAME), the inhibitor of nitric oxide (NO) synthase, as well as chemical ablation of capsaicin-sensitive afferent neurons, whereas that of 100 mM HCl was affected by neither of these treatments. We conclude that the mucosal acidification stimulates gastric HCO3- secretion in different mechanisms, depending on the concentration of acid; the response caused by 100 mM HCl is mediated only by PGs, while that caused by 200 mM HCl is mediated by both capsaicin-sensitive afferent neurons and NO, in addition to PGs.

Multiple actions of halothane on contractile response to noradrenaline in isolated mesenteric resistance arteries

Halothane, a volatile anaesthetic, produces systemic hypotension and significantly alters organ blood flow. Isometric force was recorded in isolated rat small mesenteric arteries to investigate its action on contractile response to noradrenaline, the sympathetic neurotransmitter. Halothane (1-5%) enhanced contractile response to noradrenaline in the endothelium-intact arteries, but had little influence in the endothelium-denuded arteries. However, halothane consistently inhibited the noradrenaline response in the endothelium-denuded arteries pretreated with ryanodine (10 microM). The enhancement of the contractile response to noradrenaline in the endothelium-intact arteries was unaffected by treatment with N(G)-nitro L-arginine, tetraethylammonium, apamin, charybdotoxin, indomethacin, diclofenac, nordihydroguaiaretic acid, BQ-123, BQ-788, losartan, ketanserin, or superoxide dismutase. Halothane prolonged vasorelaxation after washout of noradrenaline in the endothelium-denuded arteries. Both ryanodine and vanadate (0.1-0.3 mM), a putative inhibitor of the plasma membrane Ca2+-ATPase, also prolonged the vasorelaxation. Halothane still prolonged the vasorelaxation in the ryanodine-treated arteries, but not in the vanadate-treated arteries. Halothane decreased the pD2 value for the pCa-force relation in the beta-escin-permeabilised, endothelium-denuded arteries. Halothane appears to influence contractile response to noradrenaline through multiple actions including endothelium-dependent enhancing, endothelium-independent enhancing, and endothelium-independent inhibitory actions. Nitric oxide, endothelium-derived hyperpolarising factor, cyclooxygenase products, lipoxygenase products, endothelin-1, angiotensin-II, serotonin, and superoxide anions are not involved in the endothelium-dependent enhancement. The endothelium-independent enhancement is presumably due to its ability to stimulate Ca2+ release from the ryanodine-sensitive intracellular stores, while the endothelium-independent inhibition is due, at least in part, to depressed Ca2+-activation of contractile proteins. Halothane may inhibit the plasma membrane Ca2+-ATPase of vascular smooth muscle cells.

Characterization of the Sulfhydryl-Sensitive Site in the Enzyme Responsible for Hydrolysis of 2- Arachidonoyl-Glycerol in Rat Cerebellar Membranes

We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.

Enhancement of EFS-induced contractions, by agmatine, in guinea pig gallbladder smooth muscle strips

Electrical field stimulation of gallbladder muscle strips produces frequency-dependent contractions by activating cholinergic nerves. The cholinergic motor function of the gallbladder and enteric system is also modulated by other mediators. The aim of this study was to investigate the role of agmatine, a ligand for alpha 2-adrenoceptors and imidazoline binding sites, in the cholinergic motor activity of guinea pig gallbladder smooth muscle. Gallbladder muscle strips obtained from guinea pigs were subjected to electrical field stimulation (1-64 Hz, 100 V, 1-ms pulse width, and 10-s train duration). Frequency-response contractions of gallbladder muscle strips were traced before and after the addition of cumulative concentrations of agmatine (10(-5)-10(-3) M) to the tissue bath. The same set of experiments was repeated in the presence of different antagonists. Agmatine by itself did not produce any contractions in guinea pig gallbladder muscle strips, but significantly enhanced the contractile response produced by electrical field stimulation. Yohimbine (10(-6) M), a selective alpha 2-adrenergic blocker, neither decreased nor increased the enhancement induced by agmatine. However, idazoxan (10(-4) M), an alpha-receptor blocker and imidazoline receptor antagonist, abolished this enhanced contractile response. Pretreatment with N(W)-nitro L-arginine methyl ester (L-NAME; 30 microM), and indomethacin (10 microM) did not inhibit the effect of agmatine. Our findings indicate that agmatine has a modulator role in the electrical field stimulation-induced cholinergic contractions of guinea pig gallbladder smooth muscle strips, and this role could be mediated by imidazoline receptors. Receptor binding studies should be done to determine the presence of endogenous agmatine and imidazoline receptors in gallbladder smooth muscle.

Chronic Tumor Necrosis Factor-α Inhibition Enhances NO Modulation of Vascular Function in Estrogen-Deficient Rats

Tumor necrosis factor-alpha (TNF-alpha) is involved in the pathogenesis of vascular disease. Clinical studies have shown that postmenopausal women have higher serum TNF-alpha levels; however, whether this increase in TNF-alpha is associated with vascular dysfunction is unknown. We investigated whether estrogen deficiency is associated with increased serum TNF-alpha levels and tested the effects of in vivo TNF-alpha inhibition on vascular reactivity. Aged (12 to 15 months) Sprague-Dawley rats were ovariectomized and treated with placebo, estrogen, or a TNF-alpha inhibitor (Etanercept; 0.3 mg/kg) for 4 weeks. Serum TNF-alpha was determined by a bioassay, and vascular function was evaluated in the myograph system. Estrogen-deficient animals had higher serum levels of TNF-alpha compared with either estrogen-replaced animals or animals treated with Etanercept. Moreover, in estrogen-deficient rats, TNF-alpha inhibition reduced the constriction of mesenteric arteries to phenylephrine, increased the modulation of this vasoconstriction by the NO synthase inhibitor nitro-l-arginine methyl ester, and decreased the modulation by a superoxide scavenger (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride). Furthermore, endothelium-dependent relaxation was also enhanced by TNF-alpha antagonism. Additionally, vascular expression of endothelial NO synthase was increased in animals treated with Etanercept, whereas the expression of NAD(P)H oxidase gp91phox and p22phox subunits was decreased. These data show that estrogen-deficient female rats have higher bioactive serum TNF-alpha levels compared with estrogen-replaced animals. Moreover, a decrease in serum bioactive TNF-alpha by a soluble TNF-alpha receptor (Etanercept) results in increased modulation of vascular function by NO. These observations suggest that TNF-alpha could be a mediator of vascular dysfunction associated with estrogen deficiency.

Cardioprotective effects of Ilex paraguariensis extract: evidence for a nitric oxide-dependent mechanism

To examine the effects of an Ilex paraguariensis (Ip) extract on postischemic alterations derived from 20 min of global ischemia and 30 min of reperfusion. Isolated rat hearts were treated 10 min before ischemia and the first 10 min of reperfusion with Ip 30 microg/ml. In other hearts, chelerythrine (1 microM), a protein kinase C blocker, or l(G)-nitro l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, were administered prior to Ip infusion. Left ventricular developed pressure (LVDP), +dP/dt(max), -dP/dt(max), and left ventricular end diastolic pressure (LVEDP) were used to assess myocardial function. Thiobarbituric acid reactive substances (TBARS) were measured. Ip treatment produced an improvement of postichemic recovery (LVDP=96+/-8%; +dP/dt(max)=95+/-10%; -dP/dt(max)=90+/-12% vs. 57+/-6%, 53+/-6% and 57+/-8%, respectively, in untreated hearts) and an attenuation of the increase of LVEDP and TBARS content. Chelerythrine did not modify and l-NAME abolished the protection induced by Ip. These data are the first demonstration that Ip extract attenuates the myocardial dysfunction provoked by ischemia and reperfusion and that this cardioprotection involves a diminution of oxidative damage through a nitric oxide-dependent mechanism.