Endothelium-derived nitric oxide is involved in the hypotensive and vasorelaxant responses induced by the aqueous fraction of the ethanolic extract of the leaves of Albizia inopinata (Harms) G. P. Lewis in rats.

The acute cardiovascular effects of an aqueous fraction of the ethanolic extract of the leaves (AFL) of Albizia inopinata (Harms) G. P. Lewis (Leguminosae) were studied in rats using a combined in vivo and in vitro approach. In conscious, unrestrained rats, AFL (5, 10 and 20 mg/kg(-1) body wt. i.v., randomly) produced a significant and dose-dependent hypotension associated with increases in heart rate and cardiac output, and with a strong reduction in total peripheral resistances. The hypotensive response to AFL (20 mg/kg(-1) body wt.) was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg/kg(-1) body wt. i.v.). Furthermore, under these conditions, the associated tachycardia was inhibited completely. In isolated rat aortic rings, increasing concentrations of AFL (10, 20, 40 and 80 microg/ml(-1)) were able to antagonize the effects of phenylephrine- (1 microM) and KCl- (80 mM) induced contractions (IC50 value 65 +/- 4 and 54 +/- 6 microg/ml(-1), respectively). The smooth muscle-relaxant activity of AFL was inhibited similarly either removal of the vascular endothelium or by L-NAME (10 and 100 microM), but was not affected significantly by atropine (1 microM) or indomethacin (10 microM). In isolated rat atrial preparations, AFL (30, 100, 300 and 500 microg/ml(-1)) produced concentration-related negative inotropic and chronotropic effects (IC50 value = 274 +/- 53 and 335 +/- 23 microg/ml(-1), respectively). These results suggest that in rats, the hypotensive effect of AFL is due to a peripheral vasodilation, at least partly secondary to the release of NO by the vascular endothelium. The direct cardio-depressant actions of AFL are of little importance in the systemic effects of the extract.     

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.     

Mechanism of vasorelaxant activity of a fraction of root extract of Sesamum indicum Linn

The petroleum ether soluble fraction (SIPE) of the root extract of S. indicum was evaluated for the vasorelaxant activity using isolated rat aorta. SIPE up to 180 microg/ml concentration significantly inhibited phenylephrine- and KCl-induced contraction to the extent of 98.13 +/- 6.37 and 70.19 +/- 3.43% respectively in isolated rat aorta in a concentration dependent manner. The vasorelaxant activity was not blocked by propranolol (10 microM), atropine (1 microM) indomethacin (10 microM) and glibenclamide (10 microM). Influence of SIPE on phenylephrine-induced contractions in aortic preparations in absence of functional endothelium and on pre-incubating the tissue with L-NAME (300 microM) or methylene blue (10 microM) was also studied. SIPE at 180 microg/ml concentration could elicit partial relaxation in presence of L-NAME or methylene blue to the extent of 34.26 +/- 6.13 and 25.66 +/- 10.95% respectively. However, in absence of functional endothelium, SIPE exhibited little relaxation to the extent of 6.70 +/- 4.87%. These studies revealed that the vasorelaxant activity of SIPE was chiefly mediated through endothelium-dependent pathway.     

Calcium mobilization as the endothelium-independent mechanism of action involved in the vasorelaxant response induced by the aqueous fraction of the ethanol extract of Albizia inopinata G. P. Lewis (AFL) in the rat aorta.

In a previous work, we demonstrated that, in normotensive rats, AFL induced a marked hypotension due to a decrease in total peripheral resistances (TPR), partially secondary to the release of NO by the endothelium. NO did not, however, account for the total vasodilation produced by AFL in these rats. The aim of this study was to determine the involvement of the intracellular calcium mobilization in the vasorelaxant action induced by AFL in the rat aorta. In aorta of normotensive rats AFL (10, 20, 40 and 80 microg/ml) inhibited the sustained contractions induced by KCl (80 and 30 mM) and phenylephrine (Phe, 1 microM) with similar IC50 values (54 +/- 6, 52 +/- 4 and 65 +/- 4 microg/ml, respectively). The relaxing response induced by AFL against Phe-induced contractions was modified significantly by the endothelium removal (IC50 = 132 +/- 23 and 65 +/- 4 microg/ml, endothelium removed and intact endothelium aortic rings, respectively). Nevertheless, removal of the endothelium did not significantly change IC50 values when KCl (30 and 80 mM) was used as the contractile agent. The inhibitory effect induced by AFL on high (64.5 mM) K+-induced contraction was potentiated slightly (p < 0.05) by the decrease (from 2.5 to 0.3 mM, Ca2+) and attenuated by the increase (from 2.5 to 7.5 mM Ca2+) in the external [Ca2+]. In addition, in aortas from normotensive rats, AFL antagonized transient contractions induced in Ca2+-free media induced by 1 microM noradrenaline in a concentration-dependent manner, but not those induced by 20 mM caffeine. It is suggested that the remaining vasodilator effect of AFL in normotensive rats is probably due to an inhibition of Ca2+ influx and/or inhibition of intracellular Ca2+ mobilization from the noradrenaline-sensitive stores.     

Mechanisms of the contractile effect of the hydroalcoholic extract of Cissampelos sympodialis Eichl. in the rat aorta.

In the present work the effect of the aqueous fraction of the ethanolic extract of the leaves (AFL) of Cissampelos sympodialis Eichl. was investigated in the rat aorta. In the presence of functional endothelium, AFL produced concentration-dependent contractions (EC50 value of 76.6 +/- 17.8 micrograms/ml). In the absence of functional endothelium, the concentration-response curves to AFL were significantly shifted to the left (EC50 values of 1.3 +/- 0.9 micrograms/ml) without modification of its maximal contractile effect. In the presence of L-NAME (300 microM) and of indomethacin (10 mM), the concentration-response curves produced by AFL were also shifted to the left (EC50 values of 21.8 +/- 6.2 and 24.3 +/- 13.2 micrograms/ml, respectively). The treatment of the aortas with L-NAME (300 microM) plus indomethacin (10 microM) produced a significant shift to the left of the concentration-dependent curves of AFL (EC50 value of 4.9 +/- 2.2 micrograms/ml), similar to that observed in the absence of the vascular endothelium. In addition, AFL-induced contraction was abolished in the presence of prazosin (1 microM), and significantly shifted to the right in the presence of yohimbine (EC50 value of 723.6 +/- 76.4 micrograms/ml). Thus, based on these results, it can be concluded that contractions induced by AFL in the rat aorta were due to activation of alpha-adrenoceptors. Furthermore, these results also showed that the AFL-induced contractions were modulated by the endothelium, via the release of NO and of a cyclooxygenase-derived relaxant product. Finally, it can be concluded that the contractile effects of AFL on vascular smooth muscle may play an important role in the hypertensive effects of this plant in vivo.     

Endothelum-dependent and-independent relaxations in aortic rings obtained from hypertensive hooded (Aguti) rats.

Experimental hypertension studies are few in the hooded (Aguti) rat. The present study was designed to investigate the usefulness of this rat strain for experimental hypertension studies and to test the hypothesis that the hypertension may be associated with a diminution of endothelium dependent and independent relaxations. Hypertension was induced in inbred hooded rats (n=8 each) by administering 8% salt in the diet and /or 100 mg/kg/day Nomega-nitro-L-arginine-methyl-ester (L-NAME) in the drinking water for six and/or four weeks respectively. The rats were anaesthetized using a 25% urethane and 1% chloralose mixture given intraperitoneally at a dose of 5 mg/kg. Their blood pressure was measured invasively. Thereafter, relaxations of rat aortic preparations to acetylcholine, histamine and sodium nitroprusside (SNP) were assessed using standard organ bath conditions. Probabiliity level of 0.05 was taken as statistically significant. The mean arterial pressure (MAP;mm Hg) rose significantly in all test groups (Salt: 148.3 +/- 4.6; L-NAME: 181.7 +/-8.3; Salt+L-NAME:154.9 +/-8.7) compared with control (94.2 +/-6.8; [P < 0.05]. The MAP was significantly [P < 0.05] higher in the L-NAME group than in all the other groups. The heart rate fell significantly in the salt + L-NAME group compared to control [P <0.05].The IC50 of acetylcholine in aortic rings from L-NAME rats (7.9 x 10(-1) +/- 6.0 x 10(-3)) was significantly higher than in rings from control (9.4 x 10(-8) +/- 2.8 x 10(-8)), salt (7.8 x 10(-7) +/- 4.7 x 10(-7)) and salt + L-NAME (3.3 x 10 (-7) +/- 2.1 x 10(-7)) rats [P < 0.05]. The IC50 of histamine and SNP in the rings from the test groups of rats showed no significant difference from control. Also, endothelium dependent and independent relaxations were preserved in the various forms of hypertension studied except in chronic NOS inhibition where the former was attenuated in response to acetylcholine.     

Effects of TRIM on tension, intracellular calcium and nitrergic transmission in the rat anococcygeus muscle.

The effects of the putatively selective inhibitor of neuronal nitric oxide synthase (nNOS) 1-(2-trifluoromethylphenyl) imidazole (TRIM) were investigated on contractility, intracellular calcium and nitrergic relaxations in the rat anococcygeus muscle. TRIM (100-1000 microM) reduced the tension of rat anococcygeus muscles when contracted with guanethidine (10 microM) and clonidine (0.1 microM). Relaxations to TRIM persisted in the presence of the non-selective NOS inhibitor L-NAME (100 microM) and the inhibitor of soluble guanylate cyclase ODQ (1 microM). TRIM also reduced tension when muscles were contracted with phenylephrine (3 microM), noradrenaline (3 microM) or high K physiological salt solution (high KPSS; 60mM). Influx of calcium ([Ca(2+)](i)) in response to high KPSS was significantly reduced in the presence of TRIM (1mM). TRIM also inhibited the influx of (45)Ca(2+) induced by KPSS, but had no effect on the influx induced by phenylephrine (10 microM). TRIM (300 microM) had a modest, but significant, inhibitory effect on nitrergic relaxations that were evoked by electrical field stimulation (1-10 Hz, 15 V, 10s trains) in muscles contracted with guanethidine and clonidine. In contrast, L-NAME (1-100 microM) inhibited these nitrergic responses with an IC(50) of 9.31+/-0.87 microM (n=4). The results suggest that the smooth muscle relaxant effect of TRIM in the rat anococcygeus muscle may affect the entry of Ca(2+) possibly through voltage-operated calcium channels. Furthermore, the relatively modest effect of TRIM on nitrergic responses indicates that it is not a particularly reliable inhibitor of nNOS.     

Spasmolytic action of the methanol extract and isojuripidine from Solanum asterophorum Mart. (Solanaceae) leaves in guinea-pig ileum

Solanum asterophorum Mart. (Solanaceae) is a shrub popularly known as "jurubeba-defogo" in the northeast of Brazil. In the present work, the methanol extract (SA-MeOH, 3750 microg/mL) and isojuripidine (10(-7) - 3 x 10(-4) M), a steroidal alkaloid obtained from S. asterophorum Mart. leaves, inhibited phasic contractions induced by both 1 microM histamine [IC50 = (225.8 +/- 47.4), g/mL and (3.5 +/- 0.8) x 10(-5) M] or 1 microm acetylcholine [IC50 = (112.5 +/- 20.6) microg/mL and (2.3 +/- 0.4) x 10(-5) M] in guinea-pig ileum, respectively. The extract and isojuripidine also relaxed the ileum (SA-MeOH, 1-750 microg/mL, and isojuripidine, 10(-9) - 3 x 10(-4) M) pre-contracted with 1 M histamine [EC50 = (101.1 +/- 17.4) microg/mL and (1.2 +/- 0.3) x 10(-6) M] or 1 microM acetylcholine [EC50 = (136.8 +/- 21.1) microg/mL and (1.9 +/- 0.4) x 10(-6) M] or 40 mm KCl [EC50 = (149.4 +/- 19.5) microg/mL and (1.8 +/- 0.7) x 10(-6) M], respectively, in an equipotent and concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-operated calcium (Ca(v)) channels. To confirm this hypothesis, we evaluated their effect on cumulative CaCl2 curves in depolarizing medium nominally without Ca2+. SA-MeOH (27, 243, 500, and 750 microg/mL) and isojuripidine (3 x 10(-8), 10(-6), 3 x 10(-5), and 3 x 10(-4) M) inhibited the contractions induced by CaCl2, in a concentration-dependent manner. The concentration-response curves to CaCl2, in the presence of SA-MeOH and isojuripidine, were shifted downward in relation to a control curve in a non-parallel manner resulting in reduction of the maximum effect [E(max) = (71.2 +/- 9.2); (57.4 +/- 9.2); (43.8 +/- 3.4); (41.5 +/- 2.4) and (90.6 +/- 4.8); (74.7 +/- 8.7); (66.4 +/- 3.9); (31.3 +/- 4.1)%, respectively]. SA-MeOH and isojuripidine present spasmolytic action in guinea-pig ileum due to a partially blockade of calcium influx through Ca(v) channels.     

Microcystin-LR, a cyanobacterial toxin, induces growth inhibition and histological alterations in common reed (Phragmites australis) plants regenerated from embryogenic calli

The aim of this study was to establish the histological effects of exposure to microcystin-LR (MC-LR), a cyanotoxin, on axenic Phragmites australis plantlets. Plantlets were regenerated from embryogenic reed calli by tissue culture methods. Microcystin-LR inhibited the growth and development of embryogenic calli and the growth of reed plantlets. The 50% plantlet growth inhibitory concentration value (IC50) of MC-LR was 12 microg ml(-1) (12.07 microM) on mineral medium and 36 microg ml(-1) (36.22 microM) on Murashige-Skoog medium. In the case of roots, the IC50 value was 4.1 microg ml(-1) (4.12 microM) on both media. Microcystin-LR induced aerenchyma obturation, altered lignification of cell walls in the axial organs, root necrosis and the capture of lateral or adventitious roots in the tissues of axial organs of reed plantlets. Cyanotoxin induced the premature development of lateral roots, root coalescence and early aerenchyma formation. Our data suggest that microcystin-LR, a cyanotoxin, induced developmental and histological alterations leading to growth inhibition of reed, and the induced harms have an impact on understanding reed decay in eutrophic fresh waters.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

Platelet aggregation may not be a prerequisite for collagen-stimulated platelet generation of nitric oxide

By determining the sum of the supernatant concentrations of nitrite and nitrate the stimulated generation of nitric oxide (NO) by human washed platelets induced by a range of fibrillar collagen concentrations (0.0156-25 microg ml(-1)) was investigated. Platelet serotonin (5-hydroxytryptamine, 5-HT) efflux and platelet aggregation were also measured. Under resting conditions (0 microg ml(-1) collagen) platelet NO release was equivalent to 1.06+/-0.17 nmol per 10(8) platelets. Maximal NO release, equivalent to 2.1+/-0. 37 nmol per 10(8) platelets, was observed with only 0.0625 microg ml(-1) collagen (P<0.02, stimulated vs. resting release), higher collagen concentrations producing no further increases in platelet NO output. By contrast, maximal platelet aggregation and 5-HT efflux did not occur until collagen concentrations of 2.5 microg ml(-1) and 10-25 microg ml-1), respectively, had been achieved. L-NAME (1 mmol l(-1)) and L-NMMA (1 mmol l(-1)) inhibited stimulated platelet NO generation by 78+/-6% and 72%, respectively. Contrasting with fibrillar collagen, fibrillar beta-amyloid protein had no effect on platelet NO generation, or on 5-HT efflux or aggregation. These data perhaps indicate that NO generation by human platelets is stimulated by concentrations of fibrillar collagen insufficient to elicit an aggregatory response. Such a mechanism could operate in vivo to inhibit platelet aggregation which might otherwise be induced by low concentrations of circulating agonists.     

Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery

The present study investigated active tone development in isolated ring segments of rabbit epicardial coronary artery. Endothelium-denuded (E-) or endothelium-intact (E+) vessels treated with the NO synthase inhibitor N(omega)-nitro-L-arginine (100 microM) developed active tone, which was enhanced by stretch and reversed by the NO donor sodium nitroprusside (SNP; IC(50)=9 nM). Nifedipine abolished active tone and the contractile response to phorbol dibutyrate (PDBu; 10 nM) with the same potency (IC(50)=8 nM), whereas 300 nM PDBu responses were only partially blocked by nifedipine. The classical and novel PKC inhibitors GF-109203X (IC(50)=1-2 microM) and chelerythrine (IC(50)=4-5 microM) and the classical PKC inhibitor G?-6976 (IC(50)=0.3-0.4 microM) blocked both active tone and 10 nM PDBu responses with similar potency. Active tone development was associated with depolarization of membrane potential (E(m)) and a shift to the left of the E(m)-vs.-contraction relationship determined by varying extracellular potassium. The depolarization and leftward shift were reversed by either chelerythrine (10 microM) or SNP (30 nM). PDBu (100-300 nM) increased peak L-type calcium channel (Ca(v)) currents in isolated coronary myocytes, and this effect was reversed by chelerythrine (1 microM) or G?-6976 (200 nM). SNP (500 nM) reduced Ca(v) currents only in the presence of the PKA blocker 8-bromo-2'-O-monobutyryl-cAMPS, Rp isomer (10 microM). In conclusion, active tone development in coronary artery is suppressed by basal NO release and is dependent on both enhanced Ca(v) activity and classical PKC activity. Both E(m)-dependent and -independent processes contribute to contraction. Our results suggest that one E(m)-independent process is direct enhancement of Ca(v) current by PKC.     

The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart

The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective LOX inhibitor (NDGA) decreased control values of CF only at lower values of CPP but did not change nitrite outflow indicating antioxidant properties of NDGA. In addition, L-NAME decreased the effects induced by NDGA on CF and nitrite outflow indicating the role of NO.     

VEGF(121)- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production

The angiogenic proteins basic fibroblast growth factor (bFGF; FGF-2) and vascular endothelial growth factor 121 (VEGF(121)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(121)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(121) (10 microg x kg(-1) x day(-1), n = 12). Growth factors were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day 16, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(121)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(121)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced vascular remodeling induced by exogenous bFGF or VEGF(121) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted endothelial NO production could temper vascular remodeling in response to these angiogenic growth factors.     

Effects of N(G)-nitro-L-arginine methyl ester on endotoxin-induced leakage of lactate dehydrogenase and cytotoxicity in J774A.1 cells

In the present study, we investigated the effects of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) on tissue injury or cytotoxicity caused by endotoxin challenge by assaying lactate dehydrogenase (LDH) isozymes and cell viability in J774A.1 cells. In mice treated with L-NAME (10 mg kg(-1), i.v.), the activity of LDH in serum 18 h after endotoxin (6 mg kg(-1), i.p.) injection was not significantly different from that in mice treated with endotoxin alone. Mice injected with endotoxin exhibited leakage of LDH isozymes 3 and 5, but L-NAME did not protect against endotoxin-induced acute leakage of LDH isozymes. Treatment with L-NAME (10-1000 microM) significantly inhibited NO generation by endotoxin (1 microg ml(-1))-activated J774A.1 cells. However, L-NAME (10-1000 microM) did not affect endotoxin-induced cytotoxicity in J774A.1 cells. These findings suggested that endotoxin-induced NO formation may not contribute to tissue injury or cytotoxicity caused by endotoxin.     

The effects of nimodipine and L-NAME on coronary flow and oxidative stress parameters in isolated rat heart

The aim of this study was to assess the effects of Ca2+ channel antagonist nimodipine (in concentration which competitive inhibited phosphodiesterase 1--PDE1) on oxidative stress alone or under inhibition of nitric oxide synthase by L-NAME in isolated rat heart. The hearts from male Wistar albino rats (n=18, BM about 200 g, age 8 weeks) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure conditions (CPP, 40-120 cm H2O). The experiments were performed under control conditions, in the presence of Nimodipine (2 microM) or Nimodipine (2 microM) plus L-NAME (30 microM). Coronary flow (CF) varied in the autoregulatory range from 3.7 +/- 0.4 ml/min/g wt at 50 cm H2O to 4.35 +/- 0.79 at 90 cm H2O. Basal nitrite outflow, index of lipid peroxidation (measured as TBARS release) and superoxide anion release (O2-) (at 60 cm H2O) were 0.64 +/- 0.18 nmol/min/g wt, 0.55 +/- 0.13 micromol/min/g wt and 19.72 +/- 3.70 nmol/min/g wt, respectively. Nimodipine induced significant vasodilation at all values of CPP (from 26% at 40 cm H2O to 36% at 120 cm H2O) accompanied with significant decrease of nitrite outflow (from 59% at 40 cm H2O to 40% at 120 cm H2O), significant increase of TBARS above autoregulatory range (about 40%) and significant increase of O2- release (from 186% at 40 cm H2O to 117% at 120 cm H2O). However, perfusion with L-NAME completely reversed the effects of Nimodipine. Nimodipine-induced flow changes were decreased under L-NAME (from 3% at 40 cm H2O to 11% at 120 cm H2O) without changes in the autoregulatory range, accompanied with significantly increased nitrite outflow (from 69% at 40 cm H2O to 36% at 120 cm H2O) and TBARS release (almost 50%), as well as significantly decreased O2- release (from 50% at 40 cm H2O to 43% at 120 cm H20). Our findings show that effect of nimodipine on coronary flow should be significantly influenced by NO, TBARS and O2- release in isolated rat heart.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Involvement of G(i) proteins and Src tyrosine kinase in TNFalpha production induced by lipopolysaccharide,group B Streptococci and Staphylococcus aureus

Previous studies have suggested that heterotrimeric G(i) proteins, Src tyrosine kinase and phosphatidylinositol-3 kinase (PI3 Kinase) are involved in signaling events induced by lipopolysaccharide (LPS) leading to pro-inflammatory cytokines gene expression. To investigate the involvement of these mediators in Gram-positive bacteria induced pro-inflammatory cytokine expression, LPS (10 ng/ml), heat killed group B Streptococci (GBS 1 microg/ml) and Staphylococcus aureus (SA 10 microg/ml) were used to induce TNFalpha production in the murine J774A.1 macrophage (M?) cell line and human promonocytic THP-1 cell line. Pertussis toxin (PTx, 1 microg/ml), an inhibitor of G(i) protein; pyrazolopyrimidine-2 (PP2, 1 or 25 microM), a Src tyrosine kinase inhibitor; and LY294002 (100 nM), an inhibitor of PI3 Kinase were used to examine the involvement of G(i), Src tyrosine kinase and PI3 Kinase, respectively, in TNFalpha production. In J774A.1 cells, pretreatment with PTx and PP2 attenuated TNFalpha production induced by LPS (60+/-9% and 81+/-11% inhibition, n=3, p<0.05, respectively), GBS (95+/-1% and 80+/-6% inhibition, n=3, p<0.05, respectively) and SA (51+/-18% and 68+/-16% inhibition, n=4, p<0.05, respectively). However, pretreatment with LY 294002 inhibited LPS induced TNFalpha production (82+/-13% inhibition, n=3, p<0.05), but did not inhibit GBS or SA induced TNFalpha production. In THP-1 cells, pretreatment with PTx, PP2 and LY 294002 inhibited TNFalpha production induced by LPS (84+/-3%, 59+/-12% and 84+/-4% inhibition, n=3, p<0.05, respectively) and SA (56+/-7%, 87+/-1% and 35+/-6% inhibition, n=3, p<0.05, respectively). These data support our hypothesis that G(i)-coupled and Src tyrosine kinase-coupled signaling pathways are involved in both Gram-negative and Gram-positive bacteria induced pro-inflammatory cytokine expression. However, unlike LPS, involvement of PI3 Kinase in Gram-positive bacteria induced signaling pathways are species dependent.     

Endothelium-dependent vasodilation induced by Hancornia speciosa in rat superior mesenteric artery

The vasodilator effect of the ethanolic extract of leaves from Hancornia speciosa Gomes (HSE) was evaluated in superior mesenteric artery rings. HSE produced a concentration-dependent vasodilation (IC50 = 10.8 +/- 4.0 microg/mL) in arterial rings pre-contracted with phenylephrine, which was completely abolished in endothelium-denuded vessels. Endothelium-dependent vasodilation induced by HSE was strongly reduced by L-NAME (100 microM), a nitric oxide (NO) synthase inhibitor, but neither by atropine, a muscarinic receptor antagonist (1 microM), nor by indomethacin (10 microM), a cyclooxygenase inhibitor. In rings pre-contracted with 80 mM KCl, the vasodilator effect of HSE was shifted to the right and was completely abolished in the presence of L-NAME (100 microM). Similar effects were obtained in mesenteric rings pre-contracted with phenylephrine in the presence of KCl 25 mM alone or in addition to 100 microM L-NAME. In addition, BaCl2 (1 mM) dramatically reduced the vasodilation induced by HSE. Together, these findings led us to conclude that HSE induces an endothelium-dependent vasodilation in rat mesenteric artery, by a mechanism dependent on NO, on the activation of potassium channels and endothelium-derived hyperpolarizing factor release. Rutin, identified as a major peak in the HPLC fingerprint obtained for HSE, might contribute for the observed vasodilator effect, since it was able to induce an endothelium-dependent vasodilation in rat superior mesenteric arteries.     

Tyrosinase inhibitor isolated from the leaves of Zanthoxylum piperitum

Two flavonols, quercetin (1) and quercitrin (2), were isolated from the leaves of Zanthoxylum piperitum. Their structures were established by UV, one- and two-dimensional NMR, and mass spectroscopic methods. Quercetin showed significant inhibition against mushroom tyrosinase with an IC50 value of 3.8 microg/ml, and appeared to inhibit the polyphenol oxidase activity of tyrosinase in a competitive manner (Ki = 10 +/- 0.20 microM) when L-tyrosine was used as a substrate, although it did not inhibit the melanin production of Streptomyces bikiniensis.