Improvement of vascular function by acute and chronic treatment with the GPR30 agonist G1 in experimental diabetes mellitus

The G-protein coupled estrogen receptor 30 (GPR30) is a seven-transmembrane domain receptor that mediates rapid estrogen responses in a wide variety of cell types. This receptor is highly expressed in the cardiovascular system, and exerts vasodilatory effects. The objective of the present study was to investigate the effects of GPR30 on vascular responsiveness in diabetic ovariectomized (OVX) rats and elucidate the possible mechanism involved. The roles of GPR30 were evaluated in the thoracic aorta and cultured endothelial cells. The GPR30 agonist G1 induced a dose-dependent vasodilation in the thoracic aorta of the diabetic OVX rats, which was partially attenuated by the nitric oxide synthase (NOS) inhibitor, nitro-L-arginine methylester (L-NAME) and the GPR30-selective antagonist G15. Dose-dependent vasoconstrictive responses to phenylephrine were attenuated significantly in the rings of the thoracic aorta following the acute G1 administration in the diabetic OVX rats. This effect of G1 was abolished partially by L-NAME and G15. The acute administration of G1 increased significantly the eNOS activity and the concentration of NO in the endothelial cells exposed to high glucose. G1 treatment induced an enhanced endothelium-dependent relaxation to acetylcholine (Ach) in the diabetic OVX rats. Further examination revealed that G1 induced vasodilation in the diabetic OVX rats by increasing the phosphorylation of eNOS. These findings provide preliminary evidence that GPR30 activation leads to eNOS activation, as well as vasodilation, to a certain degree and has beneficial effects on vascular function in diabetic OVX rats.     

Effects of exercise training on the vascular reactivity of the whole kidney circulation in rabbits.

Exercise training is known to improve vasodilating mechanisms mediated by endothelium-dependent relaxing factors in the cardiac and skeletal muscle vascular beds. However, the effects of exercise training on visceral vascular reactivity, including the renal circulation, are still unclear. We used the experimental model of the isolated perfused rabbit kidney, which involves both the renal macro- and microcirculation, to test the hypothesis that exercise training improves vasodilator mechanisms in the entire renal circulation. New Zealand White rabbits were pen confined (Sed; n = 24) or treadmill trained (0% grade) for 5 days/wk at a speed of 18 m/min during 60 min over a 12-wk period (ExT; n = 24). Kidneys isolated from Sed and ExT rabbits were continuously perfused in a nonrecirculating system under conditions of constant flow and precontracted with norepinephrine (NE). We assessed the effects of exercise training on renal vascular reactivity using endothelial-dependent [acetylcholine (ACh) and bradykinin (BK)] and -independent [sodium nitroprusside (SNP)] vasodilators. ACh induced marked and dose-related vasodilator responses in kidneys from Sed rabbits, the reduction in perfusion pressure reaching 41 +/- 8% (n = 6; P < 0.05). In the kidneys from ExT rabbits, vasodilation induced by ACh was significantly enhanced to 54 +/- 6% (n = 6; P < 0.05). In contrast, BK-induced renal vasodilation was not enhanced by training [19 +/- 8 and 13 +/- 4% reduction in perfusion pressure for Sed and ExT rabbits, respectively (n = 6; P > 0.05)]. Continuous perfusion of isolated kidneys from ExT animals with N(omega)-nitro-L-arginine methyl ester (L-NAME; 300 microM), an inhibitor of nitric oxide (NO) biosynthesis, completely blunted the additional vasodilation elicited by ACh [reduction in perfusion pressure of 54 +/- 6 and 38 +/- 5% for ExT and L-NAME + ExT, respectively (n = 6; P < 0.05)]. On the other hand, L-NAME infusion did not affect ACh-induced vasodilation in Sed animals. Exercise training also increased renal vasodilation induced by SNP [36 +/- 7 and 45 +/- 10% reduction in perfusion pressure for Sed and ExT rabbits, respectively (n = 6; P < 0.05)]. It is concluded that exercise training alters the rabbit kidney vascular reactivity, enhancing endothelium-dependent and -independent renal vasodilation. This effect seems to be related not only to an increased bioavailability of NO but also to the enhanced responsiveness of the renal vascular smooth muscle to NO.     

Contributions of endothelium-derived relaxing factors to control of hindlimb blood flow in the mouse in vivo

We determined the contributions of various endothelium-derived relaxing factors to control of basal vascular tone and endothelium-dependent vasodilation in the mouse hindlimb in vivo. Under anesthesia, catheters were placed in a carotid artery, jugular vein, and femoral artery (for local hindlimb circulation injections). Hindlimb blood flow (HBF) was measured by transit-time ultrasound flowmetry. N(omega)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg plus 10 mg x kg(-1) x h(-1)), to block nitric oxide (NO) production, altered basal hemodynamics, increasing mean arterial pressure (30 +/- 3%) and reducing HBF (-30 +/- 12%). Basal hemodynamics were not significantly altered by indomethacin (10 mg x kg(-1) x h(-1)), charybdotoxin (ChTx, 3 x 10(-8) mol/l), apamin (2.5 x 10(-7) mol/l), or ChTx plus apamin (to block endothelium-derived hyperpolarizing factor; EDHF). Hyperemic responses to local injection of acetylcholine (2.4 microg/kg) were reproducible in vehicle-treated mice and were not significantly attenuated by L-NAME alone, indomethacin alone, L-NAME plus indomethacin with or without co-infusion of diethlyamine NONOate to restore resting NO levels, ChTx alone, or apamin alone. Hyperemic responses evoked by acetylcholine were reduced by 29 +/- 11% after combined treatment with apamin plus charybdotoxin, and the remainder was virtually abolished by additional treatment with L-NAME but not indomethacin. None of the treatments altered the hyperemic response to sodium nitroprusside (5 microg/kg). We conclude that endothelium-dependent vasodilation in the mouse hindlimb in vivo is mediated by both NO and EDHF. EDHF can fully compensate for the loss of NO, but this cannot be explained by tonic inhibition of EDHF by NO. Control of basal vasodilator tone in the mouse hindlimb is dominated by NO.     

Nitric oxide (NO) in normal and hypoxic vascular regulation of the spiny dogfish, Squalus acanthias

Nitric oxide (NO) is a potent vasodilator in terrestrial vertebrates, but whether vascular endothelial-derived NO plays a role in vascular regulation in fish remains controversial. To explore this issue, a study was made of spiny dogfish sharks (Squalus acanthias) in normoxia and acute hypoxia (60 min exposure to seawater equilibrated with 3% oxygen) with various agents known to alter NO metabolism or availability. In normoxia, nitroprusside (a NO donor) reduced blood pressure by 20%, establishing that vascular smooth muscle responds to NO. L-arginine, the substrate for NO synthase, had no hemodynamic effect. Acetylcholine, which stimulates endothelial NO and prostaglandin production in mammals, reduced blood pressure, but also caused marked bradycardia. L-NAME, an inhibitor of all NO synthases, caused a small 10% rise in blood pressure, but cell-free hemoglobin (a potent NO scavenger and hypertensive agent in mammals) had no effect. Acute hypoxia caused a 15% fall in blood pressure, which was blocked by L-NAME and cell-free hemoglobin. Serum nitrite, a marker of NO production, rose with hypoxia, but not with L-NAME. Results suggest that NO is not an endothelial-derived vasodilator in the normoxic elasmobranch. The hypertensive effect of L-NAME may represent inhibition of NO production in the CNS and nerves regulating blood pressure. In acute hypoxia, there is a rapid up-regulation of vascular NO production that appears to be responsible for hypoxic vasodilation.     

Supplementation of L-arginine improves hypertension and lipid metabolism but not insulin resistance in diabetic rats

Nitric oxide (NO) plays an important role in glucose and lipid metabolism. We previously reported that NO synthesis inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME), deteriorate insulin sensitivity and lipid metabolism, while the addition of L-arginine reverses this deterioration. L-arginine is a precursor of NO, and is used as a supplement in the US. In the present study, we evaluated whether the administration of L-arginine alone improves insulin resistance and serum lipid levels in insulin-resistant and hypertriglycemic rat models. Diabetic rats were divided into 3 groups: the control (Cont) group (standard diet), the L-NAME group (diet containing L-NAME), and the Arg group (diet containing L-arginine). After 4 weeks of breeding, urinary NOx, glucose infusion rate (GIR), glucose and lipid tolerance tests were performed. Urinary NOx levels were significantly lower in the L-NAME group than in the Cont group. The GIR in the L-NAME group was significantly lower than that in the Cont group, suggesting increased insulin resistance. However, the administration of L-arginine did not influence insulin resistance in the Arg group. Oral lipid administration significantly increased plasma triglyceride levels in the L-NAME group and plasma triglyceride levels were significantly lower in the Arg group than in the Cont group. The area under the curve of plasma triglyceride levels after oral lipid administration was larger in the L-NAME group than in the Cont group. The administration of L-NAME increased insulin resistance and decreased lipid metabolism. L-arginine significantly increased urinary NO secretion but did not improve insulin resistance, although it did improve lipid metabolism. These findings suggest that supplementation of L-arginine cannot improve insulin resistance in diabetic rats probably due to increased insulin secretion by L-arginine.     

Modulation of the intensity of the non-quantal transmitter release by nitric oxide (NO) at the neuromuscular junction

In the rat diaphragm muscle, nitric oxide (NO)--sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), as well as substrate for the NO synthesis L-arginine, decrease the level of hyperpolarization of the muscle fibre membrane after acetylcholine receptor blockade by the d-TC and irreversible acetylcholinesterase inhibition by armin (H-effect). Contrary to that, disruption of the NO synthesis in the muscle fibres by the NO-synthase inhibitor NG-nitrol-L-arginine methyl ester (L-NAME) results in enhancement of the H-effect both in vitro and in vivo. Inactivated SNP and inactive forms of arginine and NAME did not affect the H-effect magnitude. Haemoglobin, effectively binding the NO molecules, abolishes the suppressing effects of the SNP, SNAP and L-arginine upon the H-effect. The findings suggest that the NO could be acting as a modulator of nonquantal transmitter release at the mammalian neuromuscular junction.     

L-arginine availability determines the duration of acetylcholine-induced systemic vasodilation in vivo

In vitro studies have shown that acetylcholine-induced vasorelaxation is mediated by endothelium-derived relaxing factor/nitric oxide (EDRF/NO). EDRF/NO is synthesized from L-arginine by an enzymatic pathway that is inhibited by L-NG-methylarginine. To assess whether EDRF/NO also mediates the vasodilating action of acetylcholine in vivo, we have investigated the effect of L-arginine and L-NG-methylarginine on the hypotensive response to acetylcholine in the anesthetized guinea pig. L-arginine prolonged the duration of the depressor response to acetylcholine and L-NG-methylarginine decreased it. However, neither L-arginine nor L-NG-methylarginine modified the magnitude of acetylcholine's hypotensive effect unless the blood pressure was previously elevated by infusion with norepinephrine. Thus, de novo synthesis of nitric oxide from L-arginine contributes importantly, but not exclusively, to acetylcholine's hypotensive effect in the guinea pig. Furthermore, the concentration of circulating L-arginine may influence the duration and magnitude of acetylcholine-induced depressor responses under normotensive and hypertensive conditions.     

Effect of bis[curcumino]oxovanadium complex on non-diabetic and streptozotocin-induced diabetic rats

The effect of the vanadium complex bis[curcumino]oxovanadium (BCOV) on blood glucose level, serum lipid levels, blood pressure and vascular reactivity were studied in non-diabetic and streptozotocin-induced diabetic (STZ-diabetic) rats and compared to that of vanadyl sulfate. Blood glucose level, serum lipid levels, and blood pressure were significantly increased in STZ-diabetic rats. Vascular reactivity to various agonists such as noradrenaline and acetylcholine were significantly increased in STZ-diabetic rats. Blood glucose and serum lipid levels were restored to normal in STZ-diabetic animals treated with vanadyl sulfate at a concentration of 0.5 mmol/kg/day (p.o.). However, vanadyl sulfate at a concentration of 0.2 mmol/kg/day (p.o.) did not produce any significant change in blood glucose and lipid levels. There was no significant effect of vanadyl sulfate (0.2 or 0.5 mmol/kg/day) treatment on blood pressure and vascular reactivity in STZ-diabetic rats. Vanadyl sulfate significantly reduced the body weight of non-diabetic and STZ-diabetic rats. Moreover, it also caused severe diarrhea in both groups of animals. Treatment with BCOV (0.05, 0.1 and 0.2mmol/kg/day, p.o.) significantly decreased blood glucose level and serum lipids in STZ-diabetic rats. Furthermore, administration of BCOV to STZ-diabetic rats restored the blood pressure and vascular reactivity to agonists to normal. There was no significant change in the body weight of BCOV treated non-diabetic and STZ-diabetic rats. Diarrhea was not observed in both BCOV treated groups. In conclusion, the present study shows that the vanadium complex BCOV has antidiabetic and hypolipedimic effects. In addition, it improves the cardiovascular complications associated with diabetes.     

Nitric oxide may be required to prevent hypertension at the onset of diabetes

Nitric oxide (NO) plays an important role in the regulation of vascular tone, and evidence suggests that endothelial-dependent relaxation, possibly mediated via NO, is impaired in diabetes. However, the role of the endothelium in arterial pressure control early in diabetes, before dysfunction develops, is not known. This was evaluated in the present study by comparing the responses to induction of diabetes in vehicle-treated rats (D, n = 7) vs. rats chronically treated with N(G)-nitro-L-arginine methyl ester (L-NAME; D+L, n = 8). A nondiabetic group also was treated with L-NAME (L, n = 7) to control for L-NAME effects over time, independent of diabetes. After baseline measurements, rats were given either vehicle or L-NAME (10 microg. kg(-1). min(-1) iv) infusion throughout the experiment. Six days later, streptozotocin (60 mg/kg iv) was administered, followed by a 3-wk diabetic study period. Induction of diabetes in the D+L rats caused a marked and progressive increase in mean arterial pressure throughout the diabetic period, averaging approximately 70 mmHg greater than in the D rats and approximately 20 mmHg greater than in the L rats. Glomerular filtration rate and renal plasma flow tended to increase during diabetes, but this trend was reversed in the D+L rats. In addition, plasma renin activity increased in the D and D+L rats during week 1 of diabetes but then returned to control in the D rats, while continuing to increase in the D+L rats. These results suggest that, in the early stages of diabetes, NO synthesis is important to prevent hypertension from developing, possibly through actions to maintain glomerular filtration and suppress renin secretion.     

L-NG-monomethyl arginine inhibits the vasodilating effects of low dose of endothelin-3 on rat mesenteric arteries

We have reported that low doses of endothelin-3 (ET-3) elicited continuous vasodilation of rat mesenteric arteries, which is possibly related to endothelium-derived relaxing factor (EDRF). In order to clarify whether or not the vasodilating effects of ET-3 are associated with EDRF, we examined the effects of L-NG-monomethyl arginine (L-NMMA), an analog of L-arginine, on low-dose ET-3 induced vasodilation of rat mesente-Hc arteries. Infusion of 50 microM L-NMMA inhibited the vasodilation induced by 10(-13) M ET-3 and rather elicited an increase in perfusion pressure, which itself was decreased by infusion of 150 microM L-arginine. In the presence of 50 microM L-NMMA, 10(-13) M ET-3 did not elicit any vasodilation of the mesenteric arteries preconstricted with NE, in which 150 microM L-arginine, but not D-arginine, caused considerable vasodilation. These data suggest that the vasodilating effects of low doses of ET-3 are associated with EDRF as an endothelium-derived nitric oxide.     

Nitric oxide and the cerebral vascular function

The presence of a cholinergic vasodilator innervation to cerebral circulation is well established. Despite its high endogenous concentration in cerebral blood vessels, acetylcholine (ACh) is not the transmitter for vasodilation. This finding has led to the discovery that nitric oxide (NO), which is coreleased with ACh and neural peptides such as vasoactive intestinal polypeptide (VIP) from the respective cholinergic-nitrergic (nitric oxidergic) nerves and the VIPergic-nitrergic nerves, is the primary transmitter in relaxing smooth muscle. ACh and VIP act presynaptically to inhibit and facilitate, respectively, the release of NO. Release of NO from cerebral vascular endothelial cells is also well established. A similar system for recycling L-citrulline to L-arginine for synthesizing more NO has been demonstrated in both cerebral perivascular nerves and endothelial cells. Neuronal and endothelial NO appears to play an important role in controlling cerebral vascular tone and circulation in health and disease.     

Acetylcholine-induced vasodilation is mediated by nitric oxide and prostaglandins in human skin.

Acetylcholine (ACh) can effect vasodilation by several mechanisms, including activation of endothelial nitric oxide (NO) synthase and prostaglandin (PG) production. In human skin, exogenous ACh increases both skin blood flow (SkBF) and bioavailable NO levels, but the relative increase is much greater in SkBF than NO. This led us to speculate ACh may dilate cutaneous blood vessels through PGs, as well as NO. To test this hypothesis, we performed a study in 11 healthy people. We measured SkBF by laser-Doppler flowmetry (LDF) at four skin sites instrumented for intradermal microdialysis. One site was treated with ketorolac (Keto), a nonselective cyclooxygenase antagonist. A second site was treated with NG-nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase. A third site was treated with a combination of Keto and L-NAME. The fourth site was an untreated control site. After the three treated sites received the different inhibiting agents, ACh was administered to all four sites by intradermal microdialysis. Finally, sodium nitroprusside (SNP) was administered to all four sites. Mean arterial pressure (MAP) was monitored by Finapres, and cutaneous vascular conductance (CVC) was calculated (CVC = LDF/MAP). For data analysis, CVC values for each site were normalized to their respective maxima as effected by SNP. The results showed that both Keto and L-NAME each attenuated the vasodilation induced by exogenous ACh (ACh control = 79 +/- 4% maximal CVC, Keto = 55 +/- 7% maximal CVC, L-NAME = 46 +/- 6% maximal CVC; P < 0.05, ACh vs. Keto or L-NAME). The combination of the two agents produced an even greater attenuation of ACh-induced vasodilation (31 +/- 5% maximal CVC; P < 0.05 vs. all other sites). We conclude that a portion of the vasodilation effected by exogenous ACh in skin is due to NO; however, a significant portion is also mediated by PGs.     

Evidence of nitric oxide, a flow-dependent factor, being a trigger of liver regeneration in rats.

The hypothesis tested was that the hemodynamic consequence of partial hepatectomy (PHX) triggers the cascade of events that leads to liver regeneration. After PHX, all the portal flow must go through the remaining vascular bed, thus producing increased shear stress and release of nitric oxide (NO), which then initiates the next stages of the regeneration process. As an index of triggering of the regeneration cascade, we used an in vitro bioassay detecting the appearance of proliferating factors (PFs; various growth factors, cytokines, and hormones) in plasma 4 h after two-thirds PHX in rats. PF levels, assessed using proliferation of cultured hepatocytes, were elevated in two-thirds PHX rats, fully blocked by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), and restored by L-arginine. L-NAME inhibited liver weight restoration at 48 h but resulted in high mortality. L-NAME lacked toxic effects in non-PHX rats. NO was directly antiproliferative on cultured cells, suggesting that the proliferative effect of NO in vivo was secondary to the activation of other proliferative stimuli. The data support the hypothesis that vascular shear stress induced release of NO following PHX serves as a primary trigger to initiate the regeneration process.     

Increase of opioid mu-receptor gene expression in streptozotocin-induced diabetic rats.

Opioids play an important role in the regulation of glucose homeostasis. In the previous report, we showed that activation of opioid mu-receptors produced a plasma glucose lowering effect in diabetic rats lacking insulin. In the present study, we found that the response of opioid mu-receptor is more sensitive in streptozotocin-induced diabetic rats (STZ-diabetic rats) than in normal rats. Intravenous injection of loperamide, an agonist of opioid mu-receptors, induced a dose-dependent decrease of plasma glucose from 3 microg/kg to 60 microg/kg in fasting STZ-diabetic rats. However, loperamide decreased the plasma glucose of normal fasting rats at the doses of 0.3 mg/kg to 1.5 mg/kg, which were much higher than those needed to produce the same effect in diabetic rats. The plasma glucose-lowering action of loperamide at the dose effective in normal rats disappeared in opioid mu-receptor knockout mice, while the plasma glucose-lowering response to loperamide was still observed in wild-type mice. This opens the possibility of mediation through opioid mu-receptor in the plasma glucose-lowering action of loperamide. Moreover, the mRNA level of opioid mu-receptor in the liver markedly increased in STZ-diabetic rats compared to normal rats. Normalization of plasma glucose concentrations in STZ-diabetic rats with exogenous insulin or phlorizin reversed mRNA and protein levels of opioid mu-receptor in the liver after 4 days of treatment. This shows that correction of hyperglycemia in STZ-diabetic rats may reverse the higher gene expression of opioid mu-receptor. These results suggest that hyperglycemia is responsible for increase of opioid mu-receptor in STZ-diabetic rats.     

Blockade of nitric oxide synthase potentiates the suppression of vasodilators by norepinephrine in the hepatic artery.

We have previously shown that nitric oxide (NO) and adenosine suppress vasoconstriction induced by norepinephrine infusion and sympathetic nerve stimulation in the hepatic artery and superior mesenteric artery. NO is involved in the control of basal vascular tone in the superior mesenteric artery but not the hepatic artery. The vasodilation induced by adenosine is inhibited by NO in the superior mesenteric artery but not in the hepatic artery. Based on these known interactions of catecholamines, adenosine, and NO, the objective of this study was to test the hypothesis that NO modulates the interaction between vasoconstrictors and vasodilators in the hepatic artery. We examined the ability of norepinephrine to suppress adenosine-mediated vasodilation and the role of NO in this interaction. Hepatic arterial blood flow and pressure were monitored in pentobarbital-anesthetized cats. The maximum hepatic arterial vasoconstrictor response to norepinephrine infusion was potentiated by blockade of NO production using Nomega-nitro-L-arginine methyl ester (L-NAME), and the potentiation was reversed by L-arginine. The maximum dilator response to adenosine was only slightly suppressed (14.0+/-5.8%, P < 0.05) by norepinephrine infusion; however, after the NO blockade, the suppression by norepinephrine of the vasodilation induced by adenosine was substantially potentiated (45.2+/-9.1%, P < 0.05). Similar results were obtained for isoproterenol-induced vasodilation. We conclude that the interaction between these vasodilators and norepinephrine was modulated by NO which inhibited the vasoconstriction and the suppression of vasodilators caused by norepinephrine and that in the absence of NO production, norepinephrine-induced constriction and the ability to antagonize dilation is substantially potentiated.     

Does autonomic blockade reveal a potent contribution of nitric oxide to locomotion-induced vasodilation?

We sought to test the role of nitric oxide (NO) in governing skeletal muscle (iliac) vascular conductance during treadmill locomotion in dogs (n = 6; 3.2 and 6.4 km/h at 0% grade, and 6.4 km/h at 10% grade). As seen previously, the increase in muscle vascular conductance accompanying treadmill locomotion was little influenced by NO synthase inhibition alone with N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg iv), but the absolute value of conductance achieved during locomotion was reduced. Such ambiguous results provide an unclear picture regarding the importance of NO during locomotion. However, muscle vasodilation is normally restrained by the sympathetic system during locomotion. Thus a significant contribution by NO to the increase in vascular conductance that accompanies locomotion could be masked by partial withdrawal of the competing influence of sympathetic vasoconstrictor nerve activity secondary to the rise in arterial pressure following systemic L-NAME administration. To test this possibility, we compared the rise in muscle vascular conductance before and after L-NAME treatment while ganglionic transmission was blocked by hexamethonium. Under these conditions, L-NAME significantly reduced both the rise in vascular conductance (by 32%, P < 0.001) and the absolute level of vascular conductance (by 30%, P < 0.001) achieved during locomotion with no effect on blood flow. Thus augmented NO production normally provides a significant drive to relax vascular smooth muscle in active skeletal muscle during locomotion. Potential deficits stemming from the absence of NO following L-NAME treatment are masked by less intense sympathetic restraint when autonomic function is intact.     

Increased L-arginine uptake and inducible nitric oxide synthase activity in aortas of rats with heart failure

L-Arginine crosses the cell membrane primarily through the system y(+) transporter. The aim of this study was to investigate the role of L-arginine transport in nitric oxide (NO) production in aortas of rats with heart failure induced by myocardial infarction. Tumor necrosis factor-alpha levels in aortas of rats with heart failure were six times higher than in sham rats (P < 0.01). L-Arginine uptake was increased in aortas of rats with heart failure compared with sham rats (P < 0.01). Cationic amino acid transporter-2B and inducible (i) nitric oxide synthase (NOS) expression were increased in aortas of rats with heart failure compared with sham rats (P < 0.05). Aortic strips from rats with heart failure treated with L-arginine but not D-arginine increased NO production (P < 0.05). The effect of L-arginine on NO production was blocked by L-lysine, a basic amino acid that shares the same system y(+) transporter with L-arginine, and by the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Treatment with L-lysine and L-NAME in vivo decreased plasma nitrate and nitrite levels in rats with heart failure (P < 0.05). Our data demonstrate that NO production is dependent on iNOS activity and L-arginine uptake and suggest that L-arginine transport plays an important role in enhanced NO production in heart failure.     

Effect of centrally administered nitric oxide modulators in Brewer's yeast-induced nociception in rats

Possible modulation of Brewer's yeast-induced nociception by centrally (icv) administered nitric oxide (NO) modulators, viz., NO synthase (NOS) inhibitors, NO precursor, donors, scavengers and co-administration of NO donor (SIN-1) with NOS inhibitor (L-NAME) and NO scavenger (Hb) was investigated in rats. Administration of NOS inhibitors and NO scavenger Hb increased the pain threshold capacity significantly, whereas NO donors SIN-1, SNP and NO precursor L-arginine were found to be hyperalgesic. D-arginine, the inactive isomer of L-arginine and methylene blue, inhibitor of soluble guanylate cyclase failed to alter the nociceptive behaviour in rats. Co-administration of SIN-1 with L-NAME and Hb found to increase the nociceptive threshold. The results indicate, that centrally administered NO modulators alter the nociceptive transmission induced by Brewer's yeast in rats.     

Urotensin-II activates L-arginine/nitric oxide pathway in isolated rat aortic adventitia

Urotensin-II (U-II), a cyclic peptide widely expressed in blood vessels, has diverse vascular actions that range from potent vasoconstriction to vasodilation. Although, U-II-induced vasodilation has been shown to be partially dependent on nitric oxide (NO), the involvement of vascular adventitia-derived NO, remains unknown. The present study aimed to elucidate the activation of U-II on L-arginine/NO pathway in isolated rat aortic adventitia. In adventitia of thoracic and abdominal aortas, the l-arginine/NO pathway was similarly characterized: the uptake of l-[(3)H]arginine was Na(+)-independent, with the peak occurring over around 40 min incubation; the total NO synthase (NOS) activity was mostly calcium-independent (>90%), and significantly inhibited by a specific iNOS inhibitor AMT; the production of NO metabolites nitrate and nitrite (NO(x)) was stimulated by L-arginine but not by D-arginine. In aortic adventitia exposed to rat U-II (10(-9) and 10(-8)M) for 6 h, the V(max) of l-[(3)H]arginine uptake over 40 min incubation was significantly increased, while the K(m) of l-[(3)H]arginine uptake showed no significant change. Besides, the iNOS mRNA level was up-regulated, the total NOS activity, largely calcium-independent, was significantly induced, and the NO(x) production was significantly stimulated by U-II. According to the same protocol as U-II, the positive control lipopolysaccharide (LPS, 10 microg/ml), which had been established to activate adventitial L-arginine/NO pathway, increased l-[(3)H]arginine uptake, iNOS activity and NO(x) production to a greater extent than U-II. In addition, the total NOS activities induced by 3 and 6h incubation of U-II and LPS were significantly inhibited by a specific inhibitor of protein synthesis, actinomycin D. In conclusion, the results showed that rat U-II activated L-arginine/NOS/NO pathway in rat aortic adventitia, suggesting a potential contributive role of adventitia-derived NO in the vasodilator response of U-II.