Inhibition of nitric oxide generation unmasks vascular dysfunction in insulin-resistant,obese JCR:LA-cp rats

1. The effects of nitric oxide (NO) on vascular reactivity and platelet function in the obese (cp/cp) and lean (+/?) JCR:LA-cp rats were investigated.
2. Phenylephrine (PE; 0.1 nM–10 μM) induced contraction of isolated aortic rings in both genotypes (cp/cp and +/?) of JCR:LA-cp rats. The sensitivity to contraction with PE was enhanced in cp/cp compared with +/? rings. Rings from both genotypes showed an increased contraction upon removal of the endothelium.
3. Acetylcholine (ACh; 0.1 nM–10 μM)-induced endothelium-dependent relaxation of rings was not significantly different in the two genotypes. Both were inhibited to a similar extent by N^G-nitro-L-arginine methyl ester (L-NAME; 0.01–1 mM) when administered in vitro.
4. The nitric oxide synthase (NOS) inhibitor (L-NAME; 0.3, 1 or 3 mg ml⁻¹, p.o.) when administered in vivo increased blood pressure in cp/cp rats but not in +/? rats.
5. L-NAME resulted in greater inhibition of ACh-induced relaxation in cp/cp rings compared with +/? rings.
6. L-NAME treatment in vivo caused a decrease in cyclic GMP and NOS activity in rings from cp/cp but not +/? rats.
7. The NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 0.1 nM–10 μM)-induced relaxation of rings from +/? rats, an effect enhanced by the treatment with L-NAME in vivo.
8. Oral administration of L-NAME did not enhance the vasorelaxant effect of SNAP on rings of aorta from cp/cp animals.
9. Platelet aggregation and NOS activity were similar in both genotypes and were not modified by oral administration of L-NAME.
10. These results show that unimpaired generation of NO is crucial for maintenance of vascular tone particularly under conditions of vascular insult exemplified by insulin resistance, obesity and dyslipidemia detected in cp/cp rats.

     

Differential effect of L-NAME and S-methyl-isothiourea on leukocyte emigration in carrageenin-soaked sponge implants in rat

1. The role of nitric oxide (NO) in leukocyte (polymorphonuclear cells, monocytes and lymphocytes) emigration was studied in a model of carrageenin-sponge implants in rats.
2. The subcutaneous implantation of 1% (w/v) of λ-carrageenin-soaked sponges elicited an inflammatory response that was characterized by a time-related increase in leukocyte infiltration in the sponges and increased levels of nitrite in the exudate. Total leukocyte infiltration and nitrite production were maximal at 24 h and decreased after 48 and 96 h. The mononuclear cell influx was maximal at 48 h (21% of the total leukocytes). Therefore, this time point was used in the successive experiments.
3. Polymorphonuclear cell (PMN) and lymphocyte infiltration in the sponges significantly increased when rats were treated with the non-specific NO-synthase (NOS) inhibitor, N^G-nitro-L-arginine methylester (L-NAME) (1 mg ml⁻¹ in drinking water ad libitum). Monocyte emigration was not affected by L-NAME treatment. The nitrite levels in the exudate of L-NAME-treated rats were significantly reduced. The concomitant ingestion of L-arginine (30 mg ml⁻¹) resulted in a reversion of the L-NAME effect, while D-arginine (30 mg ml⁻¹) had no effect, indicating the involvement of the L-arginine: NO pathway.
4. Administration of L-NAME resulted also in an increased release of tumour necrosis factor-α (TNF-α) and prostacyclin (measured as the stable metabolite, 6-keto-PGF_1α). L-NAME had no effect on monocyte chemoattractant protein-1 (MCP-1) release in the exudate.
5. Since L-NAME may have effects on the local blood flow, phenylephrine (0.034 mg ml⁻² in drinking water) was used as it has an effect on the local blood flow similar to L-NAME. Phenylephrine had no effect on either leukocyte emigration, or on nitrite, TNF-α, prostacyclin or MCP-1 accumulation in the exudate.
6. In contrast, the more selective iNOS inhibitor S-methyl-isothiourea (SMT) (10 μg ml⁻¹ in drinking water) significantly reduced PMNs and lymphocyte influx in the sponge, having no effect on monocyte influx. Moreover, SMT decreased nitrite production in the exudate to a comparable extent as L-NAME.
7. Administration of SMT significantly reduced MCP-1 release in the exudate, without an effect on TNF-α or prostacyclin production. Moreover SMT did not produce any changes in local blood flow.
8. Our results show that a different outcome of the inflammatory process can be obtained depending on the types of NOS inhibitor used.

     

Investigation of the interaction between cholinergic and nitrergic neurotransmission in the pig gastric fundus

1. The interaction between the cholinergic and nitrergic innervation was investigated in circular muscle strips of the pig gastric fundus.
2. In physiological salt solution containing 4×10⁻⁶ M guanethidine, electrical field stimulation (EFS; 40 V, 0.5 ms, 0.5–32 Hz, 10 s at 4 min intervals) induced small transient relaxations at 0.5–4 Hz, and large frequency-dependent contractions, sometimes followed by off-relaxations, at 8–32 Hz.
3. In the presence of L-N^G-nitroarginine methyl ester (L-NAME; 3×10⁻⁴ M) or physostigmine (10⁻⁶ M), relaxations were reversed into contractions and contractions were enhanced. Physostigmine added to L-NAME further enhanced contractions, while addition of L-NAME to physostigmine had no additional effect. Off-relaxations were enhanced in the presence of L-NAME and physostigmine. L-NAME and physostigmine consistently increased basal tone.
4. Tissues contracted by 5-hydroxytryptamine or by acetylcholine responded to EFS in a similar way as in basal conditions and L-NAME reversed the relaxations at the lower stimulation frequencies into contractions and enhanced the contractions at the higher stimulation frequencies.
5. Off-relaxations in the presence of L-NAME were partially reduced by α-chymotrypsin (10 U ml⁻¹).
6. In the absence of physostigmine, the concentration-response curve to exogenous acetylcholine was not influenced by L-NAME.
7. Contractions of the same amplitude induced by EFS at 4 Hz and by exogenous acetylcholine were either decreased or enhanced to the same extent by sodium nitroprusside (SNP; 10⁻⁵ M), depending upon the degree of relaxation by SNP.
8. These experiments suggest that endogenous nitric oxide interferes with cholinergic neurotransmission in the pig gastric fundus by functional antagonism at the postjunctional level. The interaction is independent of the degree of contraction.

     

Role of nitric oxide in the development and partial reversal of allergen-induced airway hyperreactivity in conscious,unrestrained guinea-pigs

1. Using a conscious, unrestrained guinea-pig model of allergic asthma, we investigated the role of endogenous nitric oxide (NO) in the regulation of airway (hyper)reactivity to histamine before and after the allergen-induced early and late asthmatic reactions, by examining the effect of inhalation of the NO synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME, 12 mM, 15 min) on the histamine-induced airway obstruction of ovalbumin-sensitized guinea-pigs before, and at 5.5 h and 23.5 h after allergen challenge.
2. Before allergen challenge, inhaled L-NAME caused a significant 2.02±0.25 fold increase (P<0.01) in airway reactivity to histamine; this effect was reversed within 2.5 to 6 h after administration.
3. After the allergen-induced early asthmatic reaction at 5 h after ovalbumin provocation, a significant 3.73±0.67 fold increase (P<0.01) of the airway reactivity to histamine was observed; subsequent inhalation of L-NAME at 5.5 h had no effect on the airway hyperreactivity, reassessed at 6 h.
4. After the late asthmatic reaction, at 23 h after ovalbumin provocation, a reduced, but still significant airway hyperreactivity to histamine (2.18±0.40 fold; P<0.05) was observed. Subsequent inhalation of L-NAME now significantly potentiated the partially reduced airway hyperreactivity 1.57±0.19 fold (P<0.05) to the level observed after the early asthmatic reaction.
5. When administered 30 min before allergen exposure, L-NAME significantly enhanced the allergen-induced early asthmatic reaction. However, when administered at 5.5 h after allergen provocation, L-NAME did not affect the subsequent late asthmatic reaction.
6. These results indicate that endogenous NO is involved the regulation of histamine- and allergen-induced bronchoconstriction and that a deficiency of cNOS-derived NO contributes to the allergen-induced airway hyperreactivity to histamine after the early asthmatic reaction, while a recovery of NO deficiency may account for the partial reversal of the allergen-induced airway hyperreactivity after the late asthmatic reaction.

     

Regulation of citrulline recycling in nitric oxide-dependent neurotransmission in the murine proximal colon

1. We investigated the contribution of nitric oxide (NO) to inhibitory neuromuscular transmission in murine proximal colon and the possibility that citrulline is recycled to arginine to maintain the supply of substrate for NO synthesis.
2. Intracellular microelectrode recordings were made from circular smooth muscle cells in the presence of nifedipine and atropine (both 1 μM). Electrical field stimulation (EFS, 0.3–20 Hz) produced inhibitory junction potentials (i.j.ps) composed of an initial transient hyperpolarization (fast component) followed by a slow recovery to resting potential (slow component).
3. L-Nitro-arginine-methyl ester (L-NAME, 100 μM) selectively abolished the slow component of i.j.ps. The effects of L-NAME were reversed by L-arginine (0.2–2 mM) but not by D-arginine (2 mM). Sodium nitroprusside (an NO donor, 1 μM) reversibly hyperpolarized muscle cells. This suggests that NO mediates the slow component of i.j.ps.
4. L-Citrulline (0.2 mM) also reversed the effects of L-NAME, and this action was maintained during sustained exposures to L-citrulline (0.2 mM). This may reflect intraneuronal recycling of L-citrulline to L-arginine.
5. Higher concentrations of L-citrulline (e.g. 2 mM) had time-dependent effects. Brief exposure (15 min) reversed the effects of L-NAME, but during longer exposures (30 min) the effects of L-NAME gradually returned. In the continued presence of L-citrulline, L-arginine (2 mM) readily restored nitrergic transmission, suggesting that during long exposures to high concentrations of L-citrulline, the ability to generate arginine from citrulline was reduced.
6. Aspartate (2 mM) had no effect on i.j.ps, the effects of L-NAME, or the actions of L-citrulline in the presence of L-NAME. L-Citrulline (0.2–2 mM) alone had no effect on i.j.ps under control conditions.
7. S-methyl-L-thiocitrulline (10 μM), a novel NOS inhibitor, blocked the slow component of i.j.ps. The effects of this inhibitor were reversed by L-arginine (2 mM), but not by L-citrulline (2 mM).
8. These results suggest that i.j.ps in the murine colon result from release of multiple inhibitory neurotransmitters. NO mediates a slow component of enteric inhibitory neurotransmission. Recycling of L-citrulline to L-arginine may sustain substrate concentrations in support of NO synthesis and this pathway may be inhibited when concentrations of L-citrulline are elevated.

     

Response of normoxic pulmonary arteries of the rat in the resting and contracted state to NO synthase blockade

1. The pulmonary vasculature is normally in a low resting state of tone. It has been hypothesized that this basal tone is actively maintained by the continuous release of a vasodilator in the resting state. However, evidence for basal release of nitric oxide (NO) is inconclusive.
2. We studied the release of NO in arteries from the pulmonary circulation of male Wistar-Kyoto rats by examining the effects of the L-arginine analogue N^G-nitro-L-arginine methyl ester (L-NAME) on resting pulmonary arteries and on vessels pre-contracted with prostaglandin F_2α (PGF_2α).
3. Rats (n=21) were killed by an overdose with pentobarbitone. Pulmonary arteries were dissected (mean internal diameter 459±11 μm) and mounted in a small vessel wire myograph. Resting tensions were set to simulate transmural pressures of 17.5 mmHg.
4. L-NAME (100 μM) was found to produce a contraction of 0.64±0.09 mN mm⁻¹ in resting pulmonary arteries when added alone to the myograph bath. This contraction was not produced following removal of the endothelium. Vessel contraction to PGF_2α (100 μM) was found to be significantly greater when carried out in the presence of L-NAME (100 μM)–1.37±0.15 mN mm⁻¹ compared with 1.96±0.17 mN mm⁻¹. Dilatation following acetylcholine (ACh) (1 μM) was abolished in the presence of L-NAME (100 μM).
5. Rat pulmonary artery contraction in response to the addition of L-NAME and the absence of contraction upon removal of the endothelium provides supportive evidence of the active release of nitric oxide for the maintenance of resting tone.

     

Developmental changes in endothelium-dependent vasodilation and the influence of superoxide anions in perinatal rabbit pulmonary arteries

1. ACh-induced vasodilation was investigated in pulmonary arteries from 8 and 2 day pre-term foetal, neonatal (0–12 h and 4 day old) and adult rabbits. The effects of superoxide anion generation [with hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 15 mu ml⁻¹)], endogenous superoxide dismutase (SOD) inhibition [with the Cu-Zn SOD inhibitor triethylenetetramine (TETA, 1 mM)], endogenous superoxide anion scavenging [by superoxide dismutase (SOD, 50 u ml⁻¹)] and inhibition of endothelial nitric oxide synthase (eNOS) [with, N^ω-nitro-L-arginine methylester (L-NAME, 0.1 mM)], on basal and ACh-induced NO activity were studied by examining phenylephrine-induced contraction and ACh-induced vasodilation respectively.
2. L-NAME and endothelium removal abolished all ACh-induced vasodilation and 1 μM sodium nitroprusside fully dilated all vessels. ACh-induced vasodilation was absent in the 8 day pre-term foetus and 0–12 h neonate but present at all other ages. L-NAME itself contracted 2 day pre-term foetal vessels. At 0–12 h, SOD, but not the phosphodiesterase 5 inhibitor zaprinast (1 μM), uncovered ACh-induced vasodilation. At this age SOD reduced phenylephrine-induced contraction which was not influenced by TETA, L-NAME or HX/XO, and L-NAME itself did not cause contraction. This suggests both ACh-induced and basal NO activity are compromise in these vessels by endogenous superoxide anion production and deficiencies in endogenous SOD activity.
3. In 4 day vessels, but not adult vessels, L-NAME, TETA and HX/XO augmented contractions to phenylephrine, and L-NAME itself induced vasoconstriction, suggesting that basal NO and SOD activities were present by 4 days but were not evident in the adult. ACh-induced NO activity, and the influence of endogenous SOD on this, were present in the adult (and 4 day) vessels as superoxide generation with HX/XO significantly reduced ACh-induced vasodilation and this effect was inhibited by SOD and augmented by TETA.
4. Increased oxygen tensions >500 mmHg attenuated ACh-induced vasodilation in the foetal but not neonatal rabbits. Raising the oxygen tension from ∼20 to ∼120 mmHg revealed ACh-induced vasodilation in the 8 day pre-term vessels.
5. In summary, superoxide anion accumulation combined with deficiencies in SOD activity may transiently compromise basal and ACh-induced NO activity at birth. Experimental oxygen tensions markedly influence ACh-induced vasodilation in foetal rabbit pulmonary arteries.

     

Role of endogenous nitric oxide in allergen-induced airway responses in guinea-pigs

1. Endogenous nitric oxide (NO) can be detected in exhaled air and accumulates in inflamed airways. However its physiological role has not been fully elucidated. In this study, we investigated a role for endogenous NO in allergen-induced airway responses. Sensitised guinea-pigs were treated with N^G-nitro-L-arginine methyl ester L-NAME (2.0 mM) or aminoguanidine (AG) (2.0 mM) 30 min before the allergen challenge, and 3 and 4 h after the challenge. Alternatively, L-arginine (2.4 mM) treatment was performed 30 min before, and 2 and 3 h after the challenge. In all groups, ovalbumin (OVA) challenge (2 mg ml⁻¹ for 2 min) was performed, and airway responses, NO production, infiltration of inflammatory cells, plasma exudation and histological details were examined.
2. Allergen-challenged animals showed an immediate airway response (IAR) and a late airway response (LAR), which synchronised with an increase in exhaled NO. Treatment with L-NAME and AG did not affect IAR while they significantly blocked LAR (72% and 80% inhibition compared to vehicle) and production of NO (35% and 40% inhibition). On the other hand, treatment with L-arginine did not affect IAR but potentiated LAR (74% augmentation).
3. In bronchoalveolar lavage (BAL) fluid, allergen-induced increases in eosinophils were reduced by 48% for L-NAME treatment compared to vehicle, and increased by 56% for L-arginine treatment.
4. Treatment with L-NAME significantly decreased airway microvascular permeability to both Monastral blue (MB) and Evans blue (EB) dye (50.6% and 44% inhibition).
5. We conclude that allergen-induced LAR is closely associated with NO production, and that NO plays a critical role in inflammatory cell infiltration and plasma exudation in the allergic condition.

     

Cigarette smoke-inhibition of neurogenic bronchoconstriction in guinea-pigs in vivo: involvement of exogenous and endogenous nitric oxide

1. We investigated the effect of acute inhalation of cigarette smoke on subsequent non-adrenergic, non-cholinergic (NANC) neural bronchoconstriction in anaesthetized guinea-pigs in vivo by use of pulmonary insufflation pressure (PIP) as an index of airway tone. The contribution of endogenous nitric oxide (NO) was investigated with the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME). The contribution of plasma exudation to the response was investigated with Evans blue dye as a plasma marker.
2. Inhalation of 50 tidal volumes of cigarette smoke or air had no significant effect on baseline PIP. In the presence of propranolol and atropine (1 mg kg⁻¹ each), electrical stimulation of the vagus nerves in animals given air 30 min previously induced a frequency-dependent increase in PIP above sham stimulated controls (16 fold increase at 2.5 Hz, 24 fold increase at 10 Hz). In contrast, in smoke-exposed animals, the increase in subsequent vagally-induced PIP was markedly less than in the air controls (90% less at 2.5 Hz, 76% less at 10 Hz).
3. L-NAME (10 mg kg⁻¹), given 10 min before air or smoke, potentiated subsequent vagally-induced (2.5 Hz) NANC bronchoconstriction by 338% in smoke-exposed animals, but had no significant effect in air-exposed animals. The inactive enantiomer D-NAME (10 mg kg⁻¹) had no effect, and the potentiation by L-NAME was partially reversed by the NO-precursor L-arginine (100 mg kg⁻¹). Vagal stimulation did not affect the magnitude of vagally-induced bronchoconstriction 30 min later.
4. Cigarette smoke exposure reduced the magnitude of subsequent bronchoconstriction induced by neurokinin A (NKA) by 37% compared with the effect of NKA in air-exposed animals. L-NAME had no significant effect on the smoke-induced inhibition of NKA-induced bronchoconstriction.
5. Vagally-induced plasma exudation in the main bronchi was greater in smoke-exposed animals compared with air-exposed animals (120% greater at 2.5 Hz, 82% greater at 10 Hz).
6. We conclude that cigarette smoke-induced inhibition of subsequent NANC neurogenic bronchoconstriction is not associated with inhibition of airway plasma exudation and is mediated in part via exogenous smoke-derived NO, or another bronchoprotective molecule, and by endogenous NO.

     

Characterization of endothelium-derived relaxing factors released by bradykinin in human resistance arteries

1. Relaxing factors released by the endothelium and their relative contribution to the endothelium-dependent relaxation produced by bradykinin (BK) in comparison with different vasodilator agents were investigated in human omental resistance arteries.
2. BK produced an endothelium-dependent relaxation of arteries pre-contracted with the thromboxane A₂ agonist, U46619. The B₂ receptor antagonist, Hoe 140 (0.1, 1 and 10 μM), produced a parallel shift to the right of the concentration-response curve to BK with a pA₂ of 7.75.
3. Neither the cyclo-oxygenase inhibitor, indomethacin (10 μM) alone, the nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME, 300 μM) alone, the nitric oxide scavenger, oxyhaemoglobin (Hb, 10 μM) alone, nor the combination of L-NAME plus Hb affected the concentration-response curve to BK. Conversely, the combination of indomethacin with either L-NAME or Hb attenuated but did not abolish the BK-induced relaxation. By contrast, the relaxations produced by the Ca²⁺ ionophore, calcimycin ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187), and by the inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase, thapsigargin (THAPS), were abolished in the presence of indomethacin plus L-NAME. Also, the presence of indomethacin plus L-NAME produced contraction of arteries with functional endothelium.
4. The indomethacin plus L-NAME resistant component of BK relaxation was abolished in physiological solution (PSS) containing 40 mM KCl and vice versa. However, in the presence of KCl 40 mM, indomethacin plus L-NAME did not affect the nitric oxide donor, S-N-acetylpenicillamine-induced relaxation.
5. The indomethacin plus L-NAME resistant component of the relaxation to BK was significantly attenuated by the K⁺ channel blocker tetrabutylammonium (TBA, 1 mM). However, it was not affected by other K⁺ channel blockers such as apamin (10 μM), 4-aminopyridine (100 μM), glibenclamide (10 μM), tetraethylammonium (10 mM) and charybdotoxin (50 nM).
6. In the presence of indomethacin plus L-NAME, the relaxation produced by BK was not affected by the phospholipase A₂ inhibitor, quinacrine (10 μM) or by the inhibitor of cytochrome P450, SKF 525a (10 μM). Another cytochrome P450 inhibitor, clotrimazole (10 μM) which also inhibits K⁺ channels, inhibited the relaxation to BK.
7. These results show that BK induces endothelium-dependent relaxation in human small omental arteries via multiple mechanisms involving nitric oxide, cyclo-oxygenase derived prostanoid(s) and another factor (probably an endothelium-derived hyperpolarizing factor). They indicate that nitric oxide and cyclo-oxygenase derivative(s) can substitute for each other in producing relaxation and that the third component is not a metabolite of arachidonic acid, formed through the cytochrome P-450 pathway, in these arteries.

     

Endothelium-independent relaxation of aortic rings by the nitric oxide synthase inhibitor diphenyleneiodonium

1. The flavoprotein binder diphenyleneiodonium (DPI) is a potent, irreversible inhibitor of nitric oxide synthase (NOS), but produces only a transient pressor response following systemic administration to animals, despite evidence of persistent NOS inhibition. To characterize further the effects of DPI on vascular tone, isometric tension was recorded from rat isolated aortic rings mounted between steel wires in an organ bath.
2. The NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME, 1 mM) initiated an additional contraction of prostaglandin F_2α-preconstricted rings with endothelium which was sustained throughout the period of L-NAME exposure (+234±39% at 15 min). In contrast, addition of DPI (5 μM) to rings with endothelium produced a transient initial contraction (+111±27% at 2 min) followed by a more sustained relaxation (−27±19% at 15 min, P<0.001 vs L-NAME).
3. The contraction to DPI was also observed in rings without endothelium, was abolished by L-NAME pretreatment, and was unaffected by the α-adrenoreceptor inhibitor prazosin. Relaxation in response to DPI was not inhibited by endothelium removal or by pretreatment with either L-NAME or with the ATP-sensitive potassium channel blocker glibenclamide.
4. The endothelium-independent relaxation to DPI was inhibited at 23°C and its time course was delayed by pretreatment with the guanylate cyclase inhibitor methylene blue.
5. Thus, in addition to a transient initial contraction due to NOS inhibition, DPI produces an endothelium-independent, temperature-dependent relaxation which appears in part due to activation of guanylate cyclase. This relaxant effect of DPI may explain the transient nature of its pressor effect in vivo despite sustained NOS inhibition.

     

Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures

1. In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures.
2. Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S-nitroso-acetyl penicillamine (SNAP) or S-nitroso-L-glutathione (GSNO). Both agents (300 μM) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors.
3. Daily treatment with nitric oxide synthase inhibitors, such as N^G-nitro-L-arginine methyl ester (L-NAME, 300 μM), led to the stimulation of cell proliferation by 44±5%, n=3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth.
4. Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L-NAME was without effect.
5. The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor-γ and uncoupling protein-1, which are upregulated during differentiation.
6. Increasing cyclic GMP in cells by 8-bromo-cyclic GMP (100–1000 μM) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO-stimulated guanylyl cyclase, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), reduced the expression of peroxisome proliferator activated receptor-γ and uncoupling protein-1.

     

Scavenging of hydroxyl radicals but not of peroxynitrite by inhibitors and substrates of nitric oxide synthases

1. The nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) is widely used to study the role of NO^• in physiological and pathological processes, including its role in the generation of the cytotoxic species peroxynitrite (ONOO⁻) and of reactive oxygen radicals such as hydroxyl (OH^•). Often L-NAME is applied to tissues at mM concentrations. At such high concentrations, it might act as a free radical scavenger. A similar possibility might apply to the use of high levels of arginine to study the role of NO^. in atherogenesis.
2. We therefore examined the rate of scavenging of OH^• by L-NAME and found that L-NAME reacts more quickly with OH^. than the established ‘OH^. scavenger'' mannitol and the widely used `OH^• trap'' salicylate. However, D-NAME can scavenge OH^• at rates equal to L-NAME. Both L- and D-arginine were also good OH^• scavengers, comparable in effectiveness to mannitol.
3. Neither L-NAME, D-NAME, L-arginine nor D-arginine was able to inhibit ONOO⁻-dependent nitration of tyrosine, suggesting that they are unlikely to be scavengers of ONOO⁻-derived nitrating species.
4. Neither L-NAME, D-NAME, L-arginine nor D-arginine was able to inhibit the inactivation of α₁-antiproteinase by ONOO⁻, suggesting that they cannot prevent direct oxidations by peroxynitrite.
5. We conclude that L-NAME has sufficient activity as an OH^• scavenger to confound certain pharmacological experiments. However, this explanation of its biological effects can be ruled out if control experiments show that D-NAME has no effect and that L-arginine (also a free radical scavenger) antagonizes the action of L-NAME.

     

Involvement of nitric oxide in the inhibition of angiogenesis by interleukin-2

Interleukin-2 (IL-2), an immunoregulatory cytokine possessing antitumour activity, is an inducer of nitric oxide (NO) synthesis in mice and man. In this study, the possibility that IL-2 possesses antiangiogenic properties that account for its antitumour effects in vivo was examined. IL-2 caused a dose-dependent inhibition of angiogenesis in the chick embryo chorioallantoic membrane (CAM). This inhibition was completely reversed by the NO synthase inhibitor N^G-nitro-L-arginine methylester (L-NAME). Furthermore, IL-2 was capable of stimulating NO synthase activity in the CAM in vitro and this effect was suppressed by L-NAME. Addition of IL-2 to human umbilical vein endothelial cells (HUVECs) in culture, had no effect on their growth characteristics. These results suggest that IL-2 may be an important antiangiogenic molecule causing its effect via nitric oxide synthesis. The antiangiogenic activity of IL-2 may be, at least in part, responsible for its antitumour properties.     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

1. Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [N^ω-monomethyl-L-arginine (L-NMMA), N^ω-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1β (rhIL-1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps.
2. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na₂³⁵SO₄] and NO production by cumulated nitrite release during the period of study.
3. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1β challenge (0 to 250 U ml⁻¹ and 0 to 15 U ml⁻¹ respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1β (2500 U ml⁻¹ and 30 U ml⁻¹ respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves.
4. When studying the effect of NOS inhibitors (1 to 1000 μM) on NO production by cartilage cells stimulated with IL-1β (25 U ml⁻¹ or 5 U ml⁻¹), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L-NMMA⩾SMT>AG⩾L-NAME and (iii) AETU was cytotoxic when used in the millimolar range.
5. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1β, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME⩾L-NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range.
6. D-isomers of L-arginine analog inhibitors (1000 μM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1β treated cells. L-arginine (5000 μM) tended to reverse the correcting effect of L-NMMA (1000 μM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis.
7. Dexamethasone (0.1 to 100 μM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1β on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production.
8. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1β on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.

     

Modulation of angiotensin-converting enzyme by nitric oxide

1. The aim of the present study was to determine the effect of nitric oxide (NO) on angiotensin-converting enzyme (ACE) activity.
2. A biochemical study was performed in order to analyse the effect of the NO-donors, SIN-1 and diethylamine/NO (DEA/NO), and of an aqueous solution of nitric oxide on the ACE activity in plasma from 3-month old male Sprague-Dawley rats and on ACE purified from rabbit lung. SIN-1 significantly inhibited the activity of both enzymes in a concentration-dependent way between 1 and 100 μM. DEA/NO inhibited the activity of purified ACE from 0.1 μM to 10 μM and plasma ACE, with a lower potency, between 1 and 100 μM. An aqueous solution of NO (100 and 150 μM) also inhibited significantly the activity of both enzymes. Lineweaver-Burk plots indicated an apparent competitive inhibition of Hip-His-Leu hydrolysis by NO-donors.
3. Modulation of ACE activity by NO was also assessed in the rat carotid artery by comparing contractions elicited by angiotensin I (AI) and AII. Concentration-response curves to both peptides were performed in arteries with endothelium in the presence of the guanylyl cyclase inhibitor, ODQ (10 μM), and the inhibitor of NO formation, L-NAME (0.1 mM). NO, which is still released from endothelium in the presence of 10 μM ODQ, elicited a significant inhibition of AI contractions at low concentrations (1 and 5 nM). In the absence of endothelium, 1 μM SIN-1 plus 10 μM ODQ, as well as 10 μM DEA/NO plus 10 μM ODQ induced a significant inhibition on AI-induced contractions at 1 and 5 nM and at 1–100 nM, respectively.
4. In conclusion, we demonstrated that (i) NO and NO-releasing compounds inhibit ACE activity in a concentration-dependent and competitive way and that (ii) NO release from endothelium physiologically reduces conversion of AI to AII.

     

Alteration of flow-induced dilatation in mesenteric resistance arteries of L-NAME treated rats and its partial association with induction of cyclo-oxygenase-2

1. We investigated the response to pressure (myogenic tone) and flow of rat mesenteric resistance arteries cannulated in an arteriograph which allowed the measurement of intraluminal diameter for controlled pressures and flows. Rats were treated for 3 weeks with N^G-nitro-L-arginine methyl ester (L-NAME, 50 mg kg⁻¹ day⁻¹) or L-NAME plus the angiotensin I converting enzyme inhibitor (ACEI) quinapril (10 mg kg⁻¹ day⁻¹).
2. Mean blood pressure increased significantly in chronic L-NAME-treated rats (155±4 mmHg, n=8, vs control 121±6 mmHg, n=10; P<0.05). L-NAME-treated rats excreted significantly more dinor-6-keto prostaglandin F_1α (dinor-6-keto PGF_1α), the stable urinary metabolite of prostacyclin, than control rats. The ACEI prevented the rise in blood pressure and the rise in urinary dinor-6-keto PGF_1α due to L-NAME.
3. Isolated mesenteric resistance arteries, developed myogenic tone in response to stepwise increases in pressure (42±6 to 847±10 mN mm⁻¹, from 25 to 150 mmHg, n=9). Myogenic tone was not significantly affected by the chronic treatment with L-NAME or L-NAME+ACEI.
4. Flow (100 μl min⁻¹) significantly attenuated myogenic tone by 50±6% at 150 mmHg (n=10). Flow-induced dilatation was significantly attenuated by chronic L-NAME to 22±6% at 150 mmHg (n=10, P=0.0001) and was not affected in the L-NAME+ACEI group.
5. Acute in vitro N^G-nitro-L-arginine (L-NOARG, 10 μM) significantly decreased flow-induced dilatation in control but not in L-NAME or L-NAME+ACEI rats. Both acute indomethacin (10 μM) and acute NS 398 (cyclo-oxygenase-2 (COX-2) inhibitor, 1 μM) did not change significantly flow-induced dilatation in controls but they both decreased flow-induced dilatation in the L-NAME and L-NAME+ACEI groups. Acute Hoe 140 (bradykinin receptor inhibitor, 1 μM) induced a significant contraction of the isolated mesenteric arteries which was the same in the 3 groups.
6. Immunofluorescence analysis of COX-2 showed that the enzyme was expressed in resistance mesenteric arteries in L-NAME and L-NAME+ACEI groups but not in control. COX-1 expression was identical in all 3 groups.
7. We conclude that chronic inhibition of nitric oxide synthesis is associated with a decreased flow-induced dilatation in resistance mesenteric arteries which was compensated by an overproduction of vasodilator prostaglandins resulting in part from COX-2 expression. The decrease in flow-induced dilatation was prevented by the ACEI, quinapril.

     

Involvement of K+ channel permeability changes in the L-NAME and indomethacin resistant part of adenosine-5′-O-(2-thiodiphosphate)-induced relaxation of pancreatic vascular bed

1. We have previously demonstrated that adenosine-5′-O-(2-thiodiphosphate) (ADPβS), a potent P2Y-purinoceptor agonist, relaxed pancreatic vasculature not only through prostacyclin (PGI₂) and nitric oxide (NO) release from the endothelium but also through other mechanism(s). In this study, we investigated the effects of an inhibitor of the Na⁺/K⁺ pump, of ATP-sensitive K⁺ (K_ATP) channels and of small (SK_Ca) or large (BK_Ca) conductance Ca²⁺-activated K⁺ channels. Experiments were performed at basal tone and during the inhibition of NO synthase and cyclo-oxygenase.
2. In control conditions, ADPβS (15 μM) induced an initial transient vasoconstriction followed by a progressive and sustained vasodilatation. In the presence of N^ω-nitro-L-arginine methyl ester (L-NAME, 200 μM) the transient vasoconstriction was reversed into a one minute vasodilator effect, which was then followed by a progressive and sustained vasodilatation similar to that observed with ADPβS alone. The addition of indomethacin (10 μM) did not significantly modify the profile of ADPβS-induced vasodilatation.
3. Ouabain (100 μM) decreased basal pancreatic flow rate and did not modify ADPβS-induced relaxation. This inhibitor of the Na⁺/K⁺ pump increased the pancreatic vasoconstriction induced by L-NAME or by the co-administration of L-NAME and indomethacin. Ouabain did not modify either the L-NAME or the L-NAME/indomethacin resistant part of the ADPβS vasodilatation.
4. The K_ATP inhibitor tolbutamide (185 μM) did not significantly modify basal pancreatic flow rate and ADPβS-induced relaxation. This inhibitor which did not change L-NAME-induced vasoconstriction, significantly diminished the L-NAME resistant part of ADPβS-induced vasodilatation. Tolbutamide intensified the vasoconstriction induced by the co-administration of L-NAME and indomethacin. In contrast, the L-NAME/indomethacin resistant part of ADPβS vasodilatation was not changed by the closure of K_ATP.
5. The SK_Ca inhibitor apamin (0.1 μM) did not significantly change pancreatic vascular resistance whatever the experimental conditions (in the absence or in presence of L-NAME or L-NAME/indomethacin). In the presence of L-NAME, the closure of SK_Ca channels changed the one minute vasodilator effect of ADPβS into a potent vasoconstriction and thereafter modified only the beginning of the second part of the L-NAME-resistant part of the ADPβS-induced vasodilatation. In contrast, the L-NAME/indomethacin resistant part of ADPβS-induced relaxation remained unchanged in the presence of apamin.
6. Charybdotoxin (0.2 μM), an inhibitor of BK_Ca, increased pancreatic vascular resistance in the presence of L-NAME/indomethacin. In the presence of L-NAME, the closure of BK_Ca channels reversed the one minute vasodilator effect of ADβPS into a potent vasoconstriction and drastically diminished the sustained vasodilatation. In contrast the L-NAME/indomethacin resistant part of ADPβS-induced relaxation was not modified by the presence of charybdotoxin. Under L-NAME/indomethacin/charybdotoxin/apamin infusions, ADPβS evoked a drastic and transient vasoconstriction reaching a maximum at the second minute, which was followed by a sustained increase in the flow rate throughout the ADPβS infusion. The maximal vasodilator effect of ADPβS observed was not modified by the addition of apamin.
7. The results suggest that the L-NAME-resistant relaxation induced by ADPβS in the pancreatic vascular bed involves activation of BK_Ca, K_ATP and to a lesser extent of SK_Ca channels, but the L-NAME/indomethacin resistant part of ADPβS-induced relaxation is insensitive to the closure of K_ATP, SK_Ca and BK_Ca channels.

     

Interaction between superoxide anion and nitric oxide in the regulation of vascular endothelial function

1. Nitric oxide (NO)-mediated, endothelium-dependent vasodilator function in rat aortic smooth muscle was investigated in an in vitro model of endogenous vascular superoxide anion stress, generated by pretreatment with the Cu/Zn superoxide dismutase (SOD, EC 1.15.1.1) inhibitor, diethyldithiocarbamate (DETCA).
2. Contraction to noradrenaline (NA, 1 nM–1 μM) in endothelium-intact vessels was augmented after a 30 min pretreatment with DETCA (10 mM) followed by 30 min washout. This effect was abolished by N^G-nitro-L-arginine methyl ester (L-NAME, 0.3 mM) and removal of the endothelium and partially reversed by exogenous Cu/Zn SOD (200 u ml⁻¹).
3. Endothelium- and basal NO-dependent vasorelaxation to the phosphodiesterase (PDE) type V inhibitor ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquinazoline methanesulphonate) (0.1–10 μM) was inhibited after DETCA (10 mM) pretreatment. In addition, the ability of L-NAME (0.3 mM) to enhance established contractile tone was effectively absent.
4. In contrast, DETCA pretreatment did not significantly affect vasorelaxation to acetylcholine (ACh, 1 nM–3 μM) or S-nitroso-N-acetyl penicillamine (SNAP, 0.03–30 μM). However, L-NAME (0.3 mM) unmasked an inhibitory effect of DETCA pretreatment on vasorelaxation to SNAP in endothelium-intact vessels while markedly potentiating vasorelaxation to SNAP in control tissue.
5. L-NAME (0.3 mM)- and exogenous catalase (200 u ml⁻¹)-sensitive vasorelaxation to exogenous Cu/Zn SOD (200 u ml⁻¹) was greater after DETCA (10 mM) pretreatment in endothelium-intact aortic rings. This difference was abolished by catalase (200 u ml⁻¹).
6. In conclusion, tissue Cu/Zn SOD inhibition elicited a selective lesion in basal endothelial function in rat isolated aortic smooth muscle, consistent with the inactivation of basal NO by superoxide anion. The resulting leftward shift in nitrovasodilator reactivity, due to the loss of the tonic depression by basal NO, is likely to mask the inhibitory effect of superoxide anion on agonist-stimulated endothelial function and nitrovasodilator-derived NO, thereby accounting for the differential pattern of endothelial dysfunction after DETCA pretreatment.

     

Hypoxic dilatation of porcine small coronary arteries: role of endothelium and KATP-channels

1. The aim of the present study was to determine the cellular mechanims and potential mediators involved in hypoxic dilatation of porcine small coronary arteries.
2. Small coronary arteries were isolated from a branch of the left anterior descending artery of porcine hearts, cannulated with glass micropipettes and studied in a perfusion myograph system. At a transmural pressure of 40 mmHg, the arteries had an internal diameter of 167.8±6.6 μm (n=37).
3. In arteries contracted with acetylcholine (ACh), hypoxia (0% O₂, 30 min) caused dilatation (86.9±6.7% relaxation, n=6) in vessels with endothelium but constriction in endothelium-denuded vessels.
4. Hypoxic vasodilatation occurring in arteries with endothelium was abolished by the K_ATP channel inhibitor, glibenclamide (0.44 μM), but was not affected by inhibition of nitric oxide synthase (L-NAME, 44 μM) or cyclo-oxygenase (indomethacin, 4.4 μM).
5. Bradykinin evoked endothelium-dependent relaxation that was inhibited by L-NAME (44 μM) but not glibenclamide 0.44 μM). Cromakalim (0.1–0.3 μM), a K_ATP channel opener, caused relaxation that was inhibited by glibenclamide, but was not affected by L-NAME (44 μM) and/or indomethacin (4.4 μM).
6. Endothelium-removal inhibited vasodilatation evoked by cromakalim, but increased vasodilator responses to the NO donor, SIN-1 (10⁻⁸ to 10⁻⁵ M).
7. These results indicate that hypoxia acted directly on vascular smooth muscle of small coronary arteries to cause contraction. However, this effect was overwhelmed by endothelium-dependent relaxation in response to hypoxia. This relaxation was most likely mediated by release of an endothelium-derived factor, distinct from nitric oxide or prostacyclin, that activated smooth muscle K_ATP-channels.