Regulation of baseline vascular resistance in the canine diaphragm by nitric oxide.

1. The role played by nitric oxide (NO) in the regulation of blood flow to the canine isolated hemidiaphragm was evaluated by determining (a) the effects of the L-arginine analogues NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-L-arginine (L-NOARG), and argininosuccinic acid (ArgSA) on baseline vascular resistance and of the latter two agents on endothelium-dependent (acetylcholine, ACh) and endothelium independent (sodium nitroprusside, SNP) vasodilatation; (b) the effects of L- and D-arginine on baseline vascular resistance; and (c) the effects of L-glutamine, an inhibitor of intracellular recycling of L-citrulline to L-arginine, on baseline resistance and on the response to ACh and SNP. 2. L-NAME, L-NOARG and ArgSA (6 x 10(-4) M final concentration) increased baseline diaphragmatic vascular resistance to a similar extent (28.6 +/- 4.2%, 26.7 +/- 4.3% and 32.8 +/- 4.6% respectively). L-NOARG and ArgSA reversed the vasodilator effect of ACh but not of SNP. 3. L- and D-arginine had no effect on vascular resistance. 4. L-Glutamine (10(-3) M) increased baseline vascular resistance by 10 +/- 1.9% (P < 0.05) but did not alter responses to either ACh or SNP. 5. Basal NO release plays a role in the regulation of baseline diaphragmatic vascular resistance. L-Arginine analogues tested potently and specifically inhibited this process. Moreover, extracellular L-arginine appears to have no effect on baseline diaphragmatic vascular resistance.     

Mechanism of 5-hydroxytryptamine-induced coronary vasodilation assessed by direct detection of nitric oxide production in guinea-pig isolated heart.

1. We assessed whether a submaximal concentration (1 microM) of 5-hydroxytryptamine (5-HT) releases nitric oxide (NO) from the coronary endothelium in guinea-pig perfused heart (n = 5 or 6/group) by direct detection of NO in coronary effluent, and determined whether this accounts for the associated coronary dilation. We also tested whether saponin is a selective and specific tool for examining the role of this mechanism in mediating agonist-induced coronary dilatation. 2. Continuous 5 min perfusion with 5-HT, or acetylcholine (ACh; 1 microM), substance P (1 nM) or sodium nitroprusside (SNP; 1 microM) increased coronary flow from baseline by 3.6 +/- 0.2, 3.4 +/- 0.2, 1.8 +/- 0.1 and 4.1 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Coronary effluent NO content, detected by chemiluminescence, was correspondingly increased from baseline by 715 +/- 85, 920 +/- 136, 1019 +/- 58 and 2333 +/- 114 pmol min-1 g-1, respectively (all P < 0.05). 3. Continuous perfusion for 30 min with NG-nitro-L-arginine methyl ester (L-NAME) 100 microM reduced basal coronary effluent NO content by 370 +/- 32 pmol min-1 g-1 and coronary flow by 7.5 +/- 0.5 ml min-1 g-1 (both P < 0.05). Saponin (three cycles of 2 min of 30 micrograms ml-1 saponin perfusion interrupted by 2 min control perfusion) reduced basal coronary NO content by a similar amount (307 +/- 22 pmol min-1 g-1) but reduced basal coronary flow by only 0.6 +/- 0.2 ml min-1 g-1 (P < 0.05 versus the effect of L-NAME). 4. The increases in coronary flow in response to (5-HT), ACh and substance P were reduced (all P < 0.05) by 100 microM L-NAME to 1.2 +/- 0.3, 1.2 +/- 0.4 and 0.3 +/- 0.3 ml min-1 g-1, respectively. However, the flow increase in response to SNP was not reduced; it was in fact increased slightly to 4.8 +/- 0.4 ml min-1 g-1 (P < 0.05). 5. Similarly, after treatment with saponin, the increases in coronary flow in response to 5-HT, ACh and substance P were reduced to 2.1 +/- 0.3, 1.3 +/- 0.3 and 0.4 +/- 0.2 ml min-1 g-1, respectively (all P < 0.05). Again, the response to SNP was increased slightly to 4.6 +/- 0.5 ml min-1 g-1 (P < 0.05). 6. L-NAME and saponin also inhibited 5-HT, ACh and substance P-induced NO release (P < 0.05), without affecting equivalent responses to SNP. 7. For substance P, the change in coronary flow (delta CF) correlated with log10 delta NO in the presence and absence of saponin and L-NAME; delta CF = 1.2(log delta NO) 1.9; r = 0.92; P < 0.05. For 5-HT the relationship was delta CF = 2.2(log delta NO-2.7; r = 0.79; P < 0.05, indicating that 5-HT causes a disproportionately greater increase in coronary flow per release of NO. This was taken to indicate that 5-HT relaxes coronary vasculature in part by releasing NO, but in part by additional mechanisms. ACh resembled 5-HT in this respect. 8. Saponin had no effect on cardiac systolic or diastolic contractile function assessed by the construction of Starling curves with an isochoric intraventricular balloon. 9. In conclusion, despite its minimal effect on basal coronary flow, saponin is an effective tool for revealing endothelium-dependent actions of coronary vasodilator substances and has selectivity in that it does not impair endothelium-independent vasodilatation or cardiac contractile function. 5-HT dilates guinea-pig coronary arteries largely by the release of NO from the coronary endothelium.     

Nitric oxide and sensory nerves are involved in the vasodilator response to acetylcholine but not calcitonin gene-related peptide in rat skin microvasculature.

1. The contributions of sensory nerves and nitric oxide (NO) to vasodilator responses to acetylcholine (ACh) and calcitonin gene-related peptide (CGRP) were examined in rat skin microvasculature with a laser Doppler flowmeter to monitor relative blood flow. 2. Perfusion of ACh (100 microM; for 30 min) over a blister base on the rat hind footpad elicited microvascular vasodilatation and this response was not sustained. CGRP (1 microM; 10 min perfusion) also elicited vasodilatation and this response was maintained even when CGRP was no longer in contact with the blister base. 3. The vasodilator response to ACh was significantly smaller in rats pretreated as neonates with capsaicin to destroy primary sensory afferents than it was in age-matched controls. The vasodilator response to CGRP was unaffected by capsaicin pretreatment. 4. Selective inhibitors of NO synthase, NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA) (both at 100 microM) attenuated the vasodilator response to ACh in control rats, but had no effect on the vasodilator response to CGRP. There was a significant L-NOARG-resistant component in control rats while in capsaicin-treated rats the vasodilator response to ACh was virtually abolished by L-NOARG. The inactive stereoisomer NG-monomethyl-D-arginine (100 microM) did not affect the vasodilator response to ACh. 5. The efficacy of L-NOARG and L-NMMA as inhibitors of endothelium-dependent responses was confirmed by use of an endothelium-dependent vasodilator, the calcium ionophore A23187 (100 microM; 10 min perfusion). Vasodilatation to A23187 was strongly attenuated by both L-NOARG and L-NMMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myocardial and coronary endothelial protective effects of acetylcholine after myocardial ischaemia and reperfusion in rats: role of nitric oxide.

1. Recent experiments suggest that acetylcholine (ACh) may exert myocardial protective effects during ischaemia (I) and reperfusion (R). The present study was designed (i) to assess whether ACh limits infarct size and protects coronary endothelial cells in a rat model of I and R, (ii) to evaluate the role of ATP-sensitive potassium (KATP) channels and nitric oxide (NO) in the beneficial effect of ACh (iii) to evaluate whether the protective effect of ACh also extends to coronary endothelial cells and (iv) to assess whether ACh contributes to the beneficial effect of preconditioning. 2. Anaesthetized rats were subjected to 20 min I (left coronary artery occlusion) and 2 h of R. Infarct size was assessed by triphenyltetrazolium (TTC) staining and expressed as a % of the area at risk (India ink injection). Vascular studies were performed on 1.5-2 mm coronary segments (internal diameter 250-300 micros) removed distal to the site of occlusion and mounted in wire myographs. 3. ACh limited infarct size (from 59 +/- 3 to 26 +/- 5%, P < 0.01), and this was prevented by atropine (46 +/- 7%; P < 0.05 vs ACh), but not by the inhibitor of KATP channels, glibenclamide (29 +/- 8%). The inhibitor of NO synthesis NG-nitro L-arginine did not affect infarct size (54 +/- 5%) but abolished the beneficial effect of ACh (59 +/- 8%; P < 0.05 vs ACh), whereas the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1 limited infarct size to the same extent as ACh (28 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired endothelium-dependent relaxation in isolated resistance arteries of spontaneously diabetic rats.

1. Previous studies have shown that endothelium-dependent relaxation in the aorta of spontaneously diabetic bio bred rats (BB) is impaired. 2. We have investigated noradrenaline (NA) contractility, endothelium-dependent acetylcholine (ACh) and bradykinin (BK) relaxation, and endothelium-independent sodium nitroprusside (SNP) relaxation in mesenteric resistance arteries of recent onset BB rats and established insulin treated BB rats, compared to their age-matched non diabetic controls. 3. There was no significant difference in the maximum contractile response or sensitivity to noradrenaline in either of the diabetic groups compared to their age-matched controls. 4. Incubation with the nitric oxide synthetase inhibitor NG-nitro-L-arginine (L-NOARG) resulted in a significant increase in maximum contractile response to noradrenaline in the recent onset age-matched control group (P < 0.05). Analysis of the whole dose-response curve (using ANOVA for repeated measures with paired t test) showed a significant left-ward shift following the addition of L-NOARG (P < 0.001). A similar but less marked shift (P < 0.01) was evident in vessels from recent onset diabetics. An overall shift in both sensitivity and maximum response was also evident in the age-matched non diabetic controls of the insulin-treated group (P < 0.05). However, by contrast, there was no significant change in sensitivity in the insulin-treated diabetic rats. 5. ACh-induced endothelium-dependent relaxation was significantly impaired in the recent onset diabetic rats compared to their age-matched controls (47 +/- 11% versus 92 +/- 2%, P < 0.05, n = 6), and in the insulin treated diabetic rats (34 +/- 5% versus 75 +/- 6%, P < 0.05, n = 6). The relaxation responses to BK also were significantly impaired in the diabetic rats compared to their age-matched controls (recent onset: 20 +/- 3% versus 72 +/- 7%, P < 0.05, n = 6; insulin treated: 12 +/- 9% versus 68 +/- 7%, P < 0.05, n = 7). 6. Incubation with either the nitric oxide synthetase substrate, U-arginine, or the free radical scavenging enzyme superoxide dismutase (150 mu ml-1) failed to improve the attenuated response of acetylcholine-induced relaxation in the diabetic vessels. 7. Endothelium-dependent relaxation mediated by ACh and BK was significantly attenuated in both the diabetic and control vessels after incubation with L-NOARG. 8. Pretreatment with a cyclo-oxygenase inhibitor, indomethacin, significantly enhanced the relaxation to ACh in both the recent onset and insulin treated diabetic rats (42 +/- 10%, n = 7 versus 64 +/- 7%, n = 7, P < 0.05, and 40 +/- 5%, n = 7 versus 65 +/- 9%, n = 6, P < 0.05). 9. Following endothelium removal, there was a marked impairment in endothelium-dependent relaxation responses to ACh and BK in both the diabetic and control vessels. 10. Incubation with the thromboxane A2 receptor antagonist SQ29548, did not significantly improve the ACh endothelium-dependent relaxation response in the diabetic vessels. 11. Endothelium-independent relaxation to sodium nitroprusside was significantly impaired in the first group of diabetic vessels studied; however, subsequent studies showed no impairment of the sodium nitroprusside response in the diabetic vessels. 12. In conclusion, the ability of the endothelium to regulate vascular contractility is reduced in recent onset diabetic vessels, and significantly impaired in established insulin treated diabetics. Relaxation to the endothelium-dependent vasodilators ACh and BK was impaired in both the recent onset and the established insulin treated diabetics, and the ACh response was significantly improved following pretreatment with indomethacin, suggesting a role for a cyclo-oxygenase-derived vasoconstrictor. Preliminary studies with a thromboxane A2, receptor antagonist, SQ29548 did not significantly improve the impaired relaxation to ACh, indicating that the vasoconstrictor prostanoid is not thromboxane A2.     

The impact of subchronic cadmium poisoning on the vascular effect of nitric oxide in rats

The aim of this study was to evaluate the impact of poisoning with cadmium in hypertensive doses (50 or 200 ppm in drinking water for three months) on the basal and stimulated release NO effect in the isolated and perfused rat mesenteric bed. Mesenteric artery preparation preconstricted by norepinephrine (0.5 microg/mL) was used to determine changes in its vascular resistance induced by e-NOS synthase blocker, N-omega-nitro-L-arginine (L-NOARG) injected in increasing doses from 1.0 to 200.0 microg or acetylcholine (ACh) administered in doses from 0.05 x 10(-10) to 5.0 x 10(-10) mol before and during L-NOARG infusion (1.0 microg/mL). Vascular reactivity was measured as an increase or decrease in perfusion pressure in the constant flow system. Rats poisoned with 50 or 200 ppm of cadmium demonstrated a significant decrease (P <0.05) in vascular response to L-NOARG used in doses of 50 or 100 microg. The dose-response curve obtained for L-NOARG was shifted to the right and ED50 value was greater in the group of rats given cadmium in a dose of 200 ppm than in the controls (70.3 +/- 10.7 versus 25.7 +/- 4.8 microg, P <0.01). These rats reacted with lower expressed vasodilatation to ACh in doses to 0.2 x 10(-10) mol. In all poisoned rats, L-NOARG enhanced the effect of ACh used in doses from 0.05 to 0.5 x 10(-10) mol, whereas in the control group this effect was only achieved at 0.1 x 10(-10) mol. The serum nitric oxide concentration was decreased (P <0.05) in both groups of cadmium-treated rats. These results suggest that cadmium in hypertensive doses modifies the vascular effect of NO in basal conditions and after stimulation by ACh.     

Investigation of the contributions of nitric oxide and prostaglandins to the actions of endothelins and sarafotoxin 6c in rat isolated perfused lungs.

1. The aims of the study were to assess the contribution of prostaglandins and nitric oxide (NO) to the effects of endothelin (ETs) and sarafotoxin 6c (SX6c) in perfused rat lungs. This was carried out by using indomethacin, a cyclo-oxygenase inhibitor and NG-nitro-L-arginine (L-NOARG), a NO synthase inhibitor. Responses were studied under basal perfusion conditions and in other experiments after the elevation of vascular tone with the thromboxane-mimetic, U46619. The sub-types of ET receptors involved were characterized by use of ET receptor antagonists and cross-tachyphylaxis. 2. Pulmonary perfusion pressure (PPP), lung weight and pulmonary inflation pressure (PIP), were continuously recorded. Although L-NOARG (100 microM) did not alter basal parameters it markedly augmented the vasoconstriction and lung weight increases induced by ET-1 (50-400 pmol) or SX6C (25-200 pmol) while vasoconstrictor responses to phenylephrine were not affected by L-NOARG. 3. L-NOARG markedly potentiated the bronchoconstriction induced by ET-1 or SX6C whereas it had no effect on responses to carbachol. 4. When vascular tone was elevated, low doses (1.25-40 pmol) of ET-1, ET-3 and SX6C produced falls in PPP. The vasodilator potencies were SX6C > ET-1 = ET-3. The ETA receptor antagonist, BQ123, did not affect these depressor responses whereas the mixed ETA/ETB antagonist, bosentan, blocked them. 5. Indomethacin (10 microM) partially inhibited vasodilator response to ET-1, whereas it had no effect on SX6C-induced vasodilation. 6. L-NOARG plus indomethacin completely blocked ET-1 induced vasodilation, whereas responses to SX6C were blocked by L-NOARG alone. 7. Repeated injections of submaximal doses of ET-1 or SX6C caused tachyphylaxis to vasodilator responses. Subsequent injections of SX6C or ET-1 did not elicit depressor responses showing cross tachyphylaxis had occurred. 8. These findings indicate that under basal conditions the pulmonary vasoconstrictor, lung weight and bronchoconstrictor responses to ET-1 and SX6C are attenuated by evoked release of nitric oxide (NO). When vascular tone was elevated, lower doses of ETs and SX6C produced vasodilatation. These vasodilator responses are indirect, those to SX6C being mediated via NO production, whereas those to ET-1 involve both NO and prostanoid(s). Tachyphylaxis and ET antagonist experiments indicate that the same receptor subtype is involved in mediating the vasodilatation and that this is of the ETB type located on the endothelium. However the post-receptor vasodilator events triggered by ET-1 or SX6C appear to be different.     

L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro.

1. The effect of L-NG-nitro arginine (L-NOARG) was compared with that of L-NG-monomethyl arginine (L-NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) produced concentration-related contraction of the rabbit aorta precontracted with phenylephrine (700-900 nM). Similarly, L-NOARG (10-200 microM) and L-NMMA (30-100 microM) elevated perfusion pressure of the noradrenaline (NA, 0.6-2.5 mM)-preconstricted rat mesentery preparation. 3. L-NOARG (1.5-100 microM) and L-NMMA (3-100 microM) caused concentration-related inhibition of the vasodilator effect of ACh (0.01-1.0 microM) on the rabbit aorta without influencing responses to NP (0.03-0.5 microM). L-NOARG methyl ester (30 microM) also inhibited ACh-induced vasorelaxation with similar potency to NOARG. L-arginine (30-150 microM) but not D-arginine (100 microM) caused graded reversal of the inhibitory effect of both L-NOARG (15 microM) and L-NMMA (30 microM). Complete reversal of the effect of both inhibitors was achieved with 150 microM L-arginine. L-Alanine (50 microM), L-arginosuccinic acid (5 microM), L-citrulline (50 microM), L-methionine (50 microM) and L-ornithine (50 microM) failed to reverse the inhibitory effect of L-NOARG (15 microM). 4. L-NOARG (10-200 microM) and L-NMMA (30-100 microM) inhibited the vasodilator effect of ACh (0.006-18.0 nmol) in the rat mesentery without affecting vasodilatation due to NP (1.1-11.1 nmol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic treatment with nitric oxide synthase inhibitors on regional haemodynamic responses to vasodilators in conscious Brattleboro rats.

1. The effects of acute inhibition of nitric oxide (NO) synthase on cardiovascular responses to vasodilator challenges have already been described. We now report the responses to vasodilators during and after chronic NO synthase inhibition. 2. In conscious Brattleboro rats, the regional haemodynamic effects of 3 min infusions of acetylcholine (4 micrograms min-1), sodium nitroprusside (15 micrograms min-1) or adrenaline (0.2 micrograms min-1) were assessed (from areas under or over curves (AUC, AOC)) under control conditions, 6 and 72 h after the addition of the NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA) to the drinking water (1 mg ml-1), and 6, 24 and 48 h after the withdrawal of L-NMMA. In a separate group of Brattleboro rats, responses to acetylcholine, sodium nitroprusside and adrenaline were assessed before and 6 h after the onset of oral ingestion of the more potent nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 0.05 mg ml-1). 3. Acetylcholine caused renal vasodilation (87 +/- 11 units) and mesenteric vasoconstriction (-31 +/- 5 units), sodium nitroprusside caused vasodilatation in renal (96 +/- 12 units), mesenteric (222 +/- 13 units) and hindquarters (49 +/- 15 units) vascular beds, whereas adrenaline caused hindquarters vasodilatation (92 +/- 8 units). Seventy two h after the onset of oral ingestion of L-NMMA, acetylcholine had a decreased renal vasodilator (59 +/- 9 units) effect, sodium nitroprusside had an increased renal vasodilator (142 +/- 23 units) action, while adrenaline had a decreased hindquarters vasodilator (55 +/- 6 units) influence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of tachykinin receptors mediating plasma extravasation and vasodilatation in normal and acutely inflamed knee joints of the rat.

1. Inflammatory actions of tachykinins in normal rat knee joints were compared with those of animals with acutely inflamed joints induced by intra-articular injection of 2% carrageenan. Plasma protein extravasation in rat knee joints, measured by protein micro-turbidimetry, was induced by intra-articular perfusion of selective tachykinin receptor agonists. Changes in joint blood flow, measured by laser Doppler perfusion imaging, were produced by topical applications of selective tachykinin receptor agonists to the joint capsule. 2. Carrageenan-injected rat knee joints showed significantly higher (P < 0.001) basal plasma extravasation (56 +/- 4 micrograms ml-1, n = 5) than normal rat knee joints (10 +/- 4 micrograms ml-1, n = 6). Intra-articular perfusion of the selective neurokinin1 (NK1) receptor agonist [Sar9, Met(O2)11]-substance P (0.8 nmol min-1) for 60 min elevated the basal plasma extravasation to 90 +/- 17 micrograms ml-1 (n = 6, P < 0.001) in normal joints, and to 150 +/- 14 micrograms ml-1 (n = 5, P < 0.001) in inflamed joints. Perfusion of the selective NK1 receptor antagonist N2-[(4R)-4-hydroxy-1-(1-methyl-1H- indol-3-yl)carbonyl-L-prolyl]-N-methyl-N-phenylmethyl-3-(2-naphthyl)- L-alaninamide (FK888; 0.8 nmol min-1) for 20 min followed by co-perfusion with the NK1 receptor agonist (0.8 nmol min-1) produced complete inhibition of the NK1 receptor agonist-induced plasma extravasation in the two groups of animals (for both groups; n = 3, P < 0.001). 3. Intra-articular perfusion of the selective NK receptor agonist [Nle10]-neurokinin A4-10 (0.8 nmol min-1) and the selective NK3 receptor agonist [MePhe7]-neurokinin B (0.8 nmol min1) produced no increase in plasma extravasation in normal or in inflamed rat knee joints (n = 4 and 11, P > 0.05). 4. Topical bolus applications of the NK1 receptor agonist [Sar9, Met(O2)11]-substance P onto normal joint capsules produced dose-dependent vasodilatation expressed as a voltage increase from control level. The maximum increase in blood flow was 2.05-0.21 V from a basal voltage of 3.42 +/- 0.07 V (n = 13, P < 0.001). To a much lesser extent, administration of the NK2 receptor agonist [Nle10]-neurokinin A4-10 also produced dose-dependent vasodilatation with maximum increase of 0.46 +/- 0.08 V from a basal level of 3.38 +/- 0.1 V (n = 7, P < 0.01). Animals with acutely inflamed joints showed enhanced vasodilator responses to the NK1 and NK2 receptor agonists (for both: P vs non-inflamed joints < 0.001). Thus, the NK1 and NK2 receptor agonists produced maximum increases of 2.56 +/- 0.19 V (basal level = 5.84 +/- 0.07 V; n = 7, P < 0.001) and 1.97 +/- 0.26 V (basal level = 6.31 +/- 0.23 V; n = 11, P < 0.001), respectively. The NK3 receptor agonist [MePhe7]-neurokinin B produced no change in blood flow in normal or in inflamed rat knee joints (n = 7 and 5, P > 0.05). 5. Bolus administration of the NK1 receptor antagonist FK888 (10 pmol) alone followed 5 min later by another dose of 10 pmol FK888 (i.e. total dose of 2 x 10 pmol) applied together with the NK1 receptor selective agonist [Sar9, Met(O2)11]-substance P produced partial, but significant inhibition of the NK1 receptor agonist-induced vasodilatation in both normal (maximum response reduced by 51.9 +/- 5.4%; n = 6, P < 0.001) and inflamed rat knee joints (maximum response reduced by 49.3 +/- 6.1%; n = 5, P < 0.001). The NK2 receptor agonist [Nle10]-neurokinin A4-10-induced vasodilator responses in inflamed joints were not affected by this treatment (n = 6, P > 0.05). However, with two higher doses of FK888 (both 1 nmol), the NK1 and the NK2 receptor agonist-induced vasodilator responses were abolished in the two groups of animals (n = 6-8, P < 0.005). 6. Administration of two doses of the selective NK2 receptor antagonist (S)-N-methyl-N-[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) -butyl]benzamide (SR48968;...     

Ischaemia enhances the role of Ca2+-activated K+ channels in endothelium-dependent and nitric oxide-mediated dilatation of the rat hindquarters vasculature

1. We compared the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) and tetraethylammonium (TEA), a blocker of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, on vasodilator responses to endothelium-dependent (acetylcholine; ACh) and -independent (sodium nitroprusside; SNP) vasodilators. The mechanism of the vasodilator responses was determined in rat hindquarters under normal conditions (sham ischaemia) and after 2 h ischaemia followed by reperfusion with physiological saline. 2. In sham ischaemia, the responses to ACh were significantly reduced by L-NOARG (1 mmol/L) and TEA (1 mmol/L) and there was a further reduction in response the presence of both agents. Dilator responses to SNP were significantly enhanced by L-NOARG, whereas TEA did not alter the SNP-induced vasodilatation when given either alone or in the presence of L-NOARG. 3. After ischaemia, L-NOARG caused a similar inhibition of ACh-induced dilatation to that observed in sham ischaemia. However, TEA alone or combined with L-NOARG caused a significantly greater inhibition of the ACh-induced vasodilatation after ischaemia than observed in the sham ischaemia group. Tetraethylammonium alone did not affect the responses to SNP, but it did attenuate the enhanced dilatation observed in the presence of L-NOARG. 4. In the rat hindquarters vasculature, both nitric oxide and the opening of TEA-sensitive K(+) channels contribute to ACh-induced endothelium-dependent dilatation. In addition, a TEA-sensitive mechanism was not involved in the SNP-induced dilatation under normal conditions but, after ischaemia, if there is a further inhibition of endogenous nitric oxide by L-NOARG, exogenous nitric oxide causes dilatation that is sensitive, in part, to TEA. Thus, the contribution of the opening of BK(Ca) channels to endothelium-dependent vasodilatation assumes greater importance after ischaemia and reperfusion. This may reflect an increased ability of nitric oxide or cGMP to open BK(Ca) channels after ischaemia.     

Reduced endothelium-dependent relaxation at enhanced NO release in hearts of hypercholesterolaemic rabbits.

1. Langendorff hearts, perfused at constant volume, were prepared from rabbits fed a cholesterol-enriched diet for 4 months. Coronary perfusion pressure and nitric oxide (NO) release (oxyhaemoglobin technique) into the coronary effluent were measured continuously. Prostacyclin (PGI2) in the effluents was determined by radioimmunoassay (6-oxo-PGF1 alpha). 2. Basal NO release was not different between control and hypercholesterolaemic rabbits. However, the coronary vasculature of hypercholesterolaemic rabbits showed a considerably (> 50%) reduced endothelium-dependent relaxation in response to short-term (3 min) infusion of bradykinin (50 nM) and substance P (50 nM) (P < 0.05, n = 8-9). Under these conditions, NO release into the vessel lumen was increased, by 26%, in hypercholesterolaemic hearts (P < 0.05, n = 8-9). NG-nitro-L-arginine (L-NOARG, 30 microM) significantly attenuated both bradykinin-induced NO formation and vessel relaxation in control hearts but only NO release in hypercholesterolaemia. L-Arginine (200 microM) restored the response to that before L-NOARG but did not improve the reduced endothelium-dependent relaxation in cholesterol-fed rabbits. 3. Superoxide dismutase (10 u ml-1) significantly improved vessel relaxation without changing the hypercholesterolaemia-related coronary dysfunction. Vasodilatation in response to exogenous NO donors (linsidomine) was diminished in hypercholesterolaemia as compared to controls. 4. Basal PGI2 release was unchanged in hypercholesterolaemic hearts. There was a tendency in these hearts for greater PGI2 formation after stimulation by substance P and bradykinin (P > or = 0.05). The coronary relaxation to iloprost was unchanged. 5. The data demonstrate impaired endothelium-dependent relaxation of coronary arterial resistance vessels in hypercholesterolaemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of arginine and nitric oxide on resistance blood vessels of the perfused rat kidney.

1. The vasodilator effects of arginine, nitric oxide (NO), acetylcholine (ACh) and sodium nitroprusside (NP) in the noradrenaline-preconstricted ('high tone') perfused rat kidney have been examined. 2. L-Arginine (0.6-23 mumol) caused a biphasic change in renal perfusion pressure. D-Arginine (0.6-23 mumol) was without effect. The second vasodilator component was abolished and the first vasoconstrictor effect augmented following CHAPS-induced removal of the vascular endothelium suggesting that vasodilatation was endothelium-dependent. 3. L-Arginine salts produced transient and dose-related vasodilatation. L-Arginine methylester was the most potent with an ED50 of 2.2 +/- 0.4 mumol (n = 6). The rank order of potency of the salts tested was: methylester greater than hydroxamate greater than chloride. L-Homoarginine chloride was also vasodilator (ED50, 12.0 +/- 1.3 mumol, n = 5). D-Arginine chloride was without effect at doses up to 170 mumol. Responses to L-arginine chloride were endothelium-derived relaxing factor (EDRF)-dependent being abolished by CHAPS (4.7 mg ml-1, 30 s) and significantly inhibited (greater than 70%) by gossypol (3 microM) and nordihydroguaiaretic acid (NDGA, 10 microM). 4. Vasodilatation due to NO was unaffected by CHAPS and gossypol treatment but inhibited by NDGA. NO was approximately 3 times less potent than ACh but 3000 times more potent than L-arginine methylester. 5. Kidneys perfused for 1 h with Krebs solution containing L-arginine chloride (100 microM) or L-canavanine (50 microM) showed no change in sensitivity towards ACh or NP. Higher concentrations of L-arginine chloride (500 microM) or L-canavanine (150 microM) significantly reduced the response to both vasodilators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide synthase in dog urethra: a histochemical and pharmacological analysis.

1. To examine the presence of nitric oxide synthase (NOS) activity in female dog urethra, pharmacological experiments were performed using electrical field stimulation (EFS), guanethidine, atropine, NG-nitro-L-arginine methyl ester and L-arginine, NOS immunohistochemistry using specific anti-NOS antibody, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining were also performed. 2. EFS caused frequency-dependent contractions in all urethral preparations, but in the presence of guanethidine and atropine, EFS caused significant relaxation in the proximal urethra and was without effect on the distal urethra. 3. In the presence of guanethidine, atropine, and NG-nitro-L-arginine methyl ester, small contractions to EFS were re-established in the proximal urethra, but not in the distal urethra. NG-nitro-D-arginine methyl ester had no such effect. 4. In the presence of guanethidine, atropine, and NG-nitro-L-arginine methyl ester, the addition of L-arginine, restored the EFS-elicited relaxant responses previously seen with guanethidine and atropine alone in the proximal urethra (at 30 Hz; 12.89 +/- 5.27% to -2.44 +/- 4.43%, mean +/- s.e., P < 0.05). D-Arginine had no such effect. 5. In the distal urethra, the addition of NG-nitro-L-arginine methyl ester and then L-arginine had no effect on responses to EFS in preparations treated with guanethidine and atropine. 6. Sodium nitroprusside caused relaxation in both the proximal and distal urethra. The relaxant responses per cm2 cross sectional area in the proximal and distal urethra were 1.23 +/- 0.29, and 2.02 +/- 0.54 g cm-2 cross sectional area (mean +/- s.e.), respectively: there was no significant difference between them. 7. Both NOS and NADPH diaphorase-positive neurones were present in dog urethra, the densities of both being higher in the proximal urethra than in the distal urethra. 8. These results show that female dog urethra possesses NOS nerves and that endogenous NO may play a role in relaxation in the proximal but not the distal urethra.     

Local L-NG-monomethyl-arginine attenuates the vasodilator action of bradykinin in the human forearm.

1. Studies in animals indicate that bradykinin relaxes blood vessels directly through an action on smooth muscle and indirectly through the release of endothelium-derived mediators. Its precise mechanism of action in the human arterial circulation is not yet known. 2. In this study the effects of a specific inhibitor of nitric oxide synthase, L-NG-monomethyl-arginine (L-NMMA) and noradrenaline on the vasodilator responses to bradykinin were examined in the forearm arterial bed of healthy volunteers. Noradrenaline was used as a control for vasoconstriction by L-NMMA; glyceryl trinitrate (GTN) as a control vasodilator acting independently of the NO synthase enzyme. 3. L-NMMA (4 mumol min-1; 5 min) alone reduced resting forearm blood flow by 44% (P < 0.01; n = 6) confirming that nitric oxide plays an important role in regulating vascular tone. 4. Bradykinin (10 and 100 pmol min-1; 3 min each dose) and GTN (2 and 5 nmol min-1; 3 min each dose) increased forearm blood flow in a dose-dependent manner (percentage changes 171 +/- 17% and 398 +/- 35%, and 176 +/- 21% and 268 +/- 42%, respectively; n = 6). 5. The response to bradykinin, but not that to GTN, was attenuated by L-NMMA compared with noradrenaline (P < 0.05; n = 6), suggesting that bradykinin-induced vasodilatation in the forearm is mediated, at least in part, by stimulating release of nitric oxide.     

Modulation of vasodilatation to levcromakalim by hypoxia and EDRF in the rabbit isolated ear: a comparison with pinacidil, sodium nitroprusside and verapamil.

1. We have used an isolated buffer-perfused preparation of the rabbit ear to investigate the effects of hypoxia and inhibition of endothelium-derived relaxing factor (EDRF) synthesis on the vasodilator responses to the potassium channel opener, levcromakalim (the active (-)-enantiomer of cromakalim). The results obtained with levcromakalim have been compared with those for pinacidil, sodium nitroprusside and verapamil. 2. Levcromakalim relaxed preconstricted preparations with an EC50 = 343 +/- 41 nM and Rmax = 80.3 +/- 6.4%. Under hypoxic conditions the concentration-response curve was significantly (P < 0.01) shifted to the left with an EC50 = 118 +/- 16 nM and Rmax = 89.9 +/- 2.7%. Hypoxia did not influence relaxation to either pinacidil, sodium nitroprusside or verapamil. 3. Inhibition of EDRF synthesis with 100 microM NG-nitro-L-arginine methyl ester (L-NAME) also significantly (P < 0.001) increased the vasodilator potency of levcromakalim (EC50 = 56 +/- 5 nM), and caused a similar shift in the concentration-response curve to sodium nitroprusside. It did not influence vasodilation to either verapamil or pinacidil. The potentiation of vasodilator responses to levcromakalim by L-NAME was reversed by an excess of L-arginine. 4. Impairment of oxidative phosphorylation with 400 nM carbonyl cyanide m-chlorophenylhydrazone significantly (P < 0.05) increased the potency of levcromakalim (EC50 = 120 +/- 20 nM) but did not influence vasodilation to pinacidil or endothelium-dependent relaxations to acetylcholine. 5. Vasodilatation to levcromakalim was augmented both by hypoxia and by inhibition of EDRF activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transhepatic action of ATP on the hepatic arterial and portal venous vascular beds of the rabbit: the role of nitric oxide.

1. The effect of bolus administration of adenosine 5'-triphosphate (ATP) into the portal vein on hepatic arterial pressure (the transhepatic action of ATP) and portal venous pressure, and the contribution of nitric oxide towards these responses, was studied in the in vitro dual-perfused rabbit liver. 2. At basal tone, hepatic arterial and portal venous vasoconstriction followed ATP injection, while at a tone raised with methoxamine (10(-6)-10(-5) M) ATP caused hepatic arterial vasodilatation, and a phasic vasodilatation followed by vasoconstriction in the portal venous vascular bed. 3. To determine whether the transhepatic arterial dilatation was due to the diffusion of nitric oxide (NO) from the portal venous vasculature, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), an inhibitor of NO synthesis, was infused selectively into the portal vein. L-NAME infusion potentiated portal venous vasoconstriction to ATP (-log M ED50 5.32 +/- 0.31 to 6.51 +/- 0.43, P < 0.05, Student's paired t test) indicating the possible inhibition of a NO-mediated vasodilator component of the portal venous response to ATP. There was, however, no demonstrable difference in the transhepatic arterial vasodilatation induced by ATP during this infusion. 4. Simultaneous perfusion of both the hepatic arterial and portal venous inflows with L-NAME (100 microM) resulted in a significant decrease in the amplitude of hepatic arterial responses to ATP demonstrating that these responses were ultimately mediated by an NO-dependent mechanism. 5. This study has thus demonstrated a vasodilator component of the portal venous response to ATP that is NO-mediated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of nitric oxide to beta2-adrenoceptor mediated vasodilatation in human forearm arterial vasculature

AIMS: beta2-adrenoceptor agonists are generally considered to produce endothelium independent vasodilatation through adenylate cyclase. We determined whether nitric oxide contributes to beta2-adrenoceptor vasodilatation in human arterial vasculature. METHODS: Forearm blood flow responses to brachial intra-arterial infusions of ritodrine (2.5-50 microg min(-1)), a selective beta2-adrenoceptor agonist, were determined in 24 healthy, normotensive subjects (mean age 22 years, 5F) on two occasions with initial and concomitant administration of L-NMMA (800 microg min(-1)), an NO synthase inhibitor, or noradrenaline (5-30 ng min(-1)), a control constrictor not affecting basal NO activity. Responses to the endothelium dependent vasodilator scrotonin (n = 6) and an endothelium independent vasodilator GTN (n = 9) were also determined. RESULTS: Maximal dilatation to ritodrine during L-NMMA infusion (310+/-32%; mean+/-s.e.mean) was reduced compared to that during noradrenaline infusion (417+/-41%, P<0.05), as were summary responses (1023+/-101 vs 1415+/-130; P<0.05). Responses to GTN were unaffected by L-NMMA compared to noradrenaline; max 177+/-26 vs 169+/-20%, 95% CI for difference -33,48; P=0.68; summary response 361+/-51 vs 396+/-37, 95% CI -142,71; P=0.46. Dilator responses to serotonin were reduced by L-NMMA; max 64+/-20 vs 163+/-26%, P<0.01; summary response 129+/-36 vs 293+/-60; P<0.05) and to a greater extent than ritodrine (58+/-7 vs 25+/-14%, P<0.05). CONCLUSIONS: beta2-adrenoceptor mediated vasodilatation in the human forearm has an NO mediated component. The underlying mechanism for this effect is unclear, but flow mediated vasodilatation is unlikely to be responsible.     

Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in acetylcholine relaxation of isolated human small arteries

The main purpose of the study was to clarify to which extent nitric oxide (NO) contributes to acetylcholine (ACh) induced relaxation of human subcutaneous small arteries. Arterial segments were mounted in myographs for recording of isometric tension, NO concentration and smooth muscle membrane potential. In noradrenaline-contracted arteries, ACh induced endothelium-dependent relaxations. The NO synthase inhibitor, N(G)-nitro-L-arginine (L-NOARG) had a small significant effect on the concentration-response curves for ACh, and in the presence of L-NOARG, indomethacin only caused a small additional rightward shift in the ACh relaxation. The NO scavenger, oxyhaemoglobin attenuated relaxations for ACh and for the NO donor S-nitroso-N-acetylpenicillamine (SNAP). Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), and inhibition of protein kinase G with beta-phenyl-1, N2-etheno-8-bromoguanosine- 3', 5'- cyclic monophosphorothioate, Rp-isomer, slightly attenuated ACh relaxation, but abolished SNAP induced relaxation. ACh induced relaxation without increases in the free NO concentration. In contrast, for equivalent relaxation, SNAP increased the NO concentration 32+/-8 nM. ACh hyperpolarized the arterial smooth muscle cells with 11.4+/-1.3 mV and 10.5+/-1.3 mV in the absence and presence of L-NOARG, respectively. SNAP only elicited a hyperpolarization of 1.6+/-0.9 mV. In the presence of indomethacin and L-NOARG, ACh relaxation was almost unaffected by lipoxygenase inhibition with nordihydroguaiaretic acid, or cytochrome P450 inhibition with 17-octadecynoic acid or econazole. ACh relaxation was strongly reduced by the combination of charybdotoxin and apamin, but small increments in the extracellular potassium concentration induced no relaxations. The study demonstrates that the NO/L-arginine pathway is present in human subcutaneous small arteries and to a limited extent is involved in ACh induced relaxation. The study also suggests a small contribution of arachidonic acid metabolites. However, ACh relaxation is mainly dependent on a non-NO, non-prostanoid endothelium dependent hyperpolarization. British Journal of Pharmacology (2000) 129, 184 - 192     

Bradykinin and changes in microvascular permeability in the hamster cheek pouch: role of nitric oxide.

1. The objective of this study in the hamster cheek pouch was to investigate the role of nitric oxide in bradykinin-induced microvascular leakage. The cheek pouch microcirculatory bed of the anaesthetized hamster was directly observed under microscope and vascular leakage was evidenced by dextranfluorescein isothiocyanate (FITC-dextran) extravasation. 2. Bradykinin superfusion (but not [des-Arg9]-bradykinin up to 3 x 10(-6) M) induced an increase in microvascular permeability (log EC50: -6.5 +/- 0.4) which was exclusively located on the post-capillary venule. Plasma extravasation was blocked by intravenous pretreatment with Hoe 140, a bradykinin B2 receptor antagonist (estimated log ID50: -9.5 +/- 0.2). 3. The effects of bradykinin (3 x 10(-7) M) superfusion were partially but significantly inhibited by indomethacin (10(-5) M, P < 0.05) and abolished by pretreatment with L-nitro-arginine (L-NOARG; 10(-5) M). 4. Acetylcholine (10(-6) M, which releases endothelial nitric oxide (NO), and sodium nitroprusside (10(-6) M, a nitrovasodilator) superfusion did not induce any changes in permeability, per se. Cromakalim (10(-5) M, a potassium channel opener) superfusion induced a moderate but significant plasma extravasation. 5. The effects of bradykinin, blocked by L-NOARG pretreatment, were restored by the co-perfusion of either sodium nitroprusside or cromakalim. Conversely vasoconstriction, produced by a stable analogue of thromboxane A2 (U46619, 3 x 10(-7) M), inhibited the increase in permeability produced by bradykinin. 6. The measurement of arteriolar diameter showed that bradykinin induced a vasodilatation which was blocked by L-NOARG. L-NOARG in itself was a powerful vasoconstrictor. Sodium nitroprusside and cromakalim, in the presence of L-NOARG, were able to restore the inhibited vasodilator response to bradykinin. 7. These results suggest: (1) bradykinin-induced microvascular leakage is mediated by bradykinin B2 receptor activation; (2) the increase in permeability is due to two different independent phenomena, i.e. post-capillary venular endothelial gap formation and arteriolar vasodilatation which increases the post-capillary venular transmural pressure: (3) NO is only involved in the arteriolar dilatation component of the bradykinin-induced increase in microvascular permeability.