Friedrich Brunner

Inhibition of nitric oxide synthesis by methylene blue

Methylene blue appears to inhibit nitric oxide-stimulated soluble guanylyl cyclase and has been widely used for inhibition of cGMP-mediated processes. We report here that endothelium-dependent relaxation of isolated blood vessels and NO synthase-dependent cGMP formation in cultured endothelial cells were both markedly more sensitive to inhibition by methylene blue than effects induced by direct activation of soluble guanylyl cyclase. These discrepancies were also observed when superoxide dismutase (SOD) was present to protect NO from inactivation by superoxide anion. Subsequent experiments showed that formation of L-citrulline by purified NO synthase was completely inhibited by 30 microM methylene blue (IC50 = 5.3 and 9.2 microM in the absence and presence of SOD, respectively), whereas guanylyl cyclase stimulated by S-nitrosoglutathione was far less sensitive to the drug (50% inhibition at approximately 60 microM, and maximal inhibition of 72% at 1 mM methylene blue). Experimental evidence indicated that oxidation of NADPH, tetrahydrobiopterin or reduced flavins does not account for the inhibitory effects of methylene blue. Our data suggest that methylene blue acts as a direct inhibitor of NO synthase and is a much less specific and potent inhibitor of guanylyl cyclase than hitherto assumed.

Inhibition of nitric oxide synthesis by NG‐nitro‐L‐arginine methyl ester (L‐NAME): requirement for bioactivation to the free acid, NG‐nitro‐L‐arginine

1 The L‐arginine derivatives NG‐nitro‐L‐arginine (L‐NOARG) and NG‐nitro‐L‐arginine methyl ester (L‐NAME) have been widely used to inhibit constitutive NO synthase (NOS) in different biological systems. This work was carried out to investigate whether L‐NAME is a direct inhibitor of NOS or requires preceding hydrolytic bioactivation to L‐NOARG for inhibition of the enzyme. 2 A bolus of L‐NAME and L‐NOARG (0.25 μmol) increased coronary perfusion pressure of rat isolated hearts to the same extent (21 ± 0.8 mmHg; n = 5), but the effect developed more rapidly following addition of L‐NOARG than L‐NAME (mean half‐time: 0.7 vs. 4.2 min). The time‐dependent onset of the inhibitory effect of L‐NAME was paralleled by the appearance of L‐NOARG in the coronary effluent. 3 Freshly dissolved L‐NAME was a 50 fold less potent inhibitor of purified brain NOS (mean IC50 = 70 μm) than L‐NOARG (IC50 = 1.4 μm), but the apparent inhibitory potency of L‐NAME approached that of L‐NOARG upon prolonged incubation at neutral or alkaline pH. H.p.l.c. analyses revealed that NOS inhibition by L‐NAME closely correlated with hydrolysis of the drug to L‐NOARG. 4 Freshly dissolved L‐NAME contained 2% of L‐NOARG and was hydrolyzed with a half‐life of 365 ± 11.2 min in buffer (pH 7.4), 207 ± 1.7 min in human plasma, and 29 ± 2.2 min in whole blood (n = 3 in each case). When L‐NAME was preincubated in plasma or buffer, inhibition of NOS was proportional to formation of L‐NOARG, but in blood the inhibition was much less than expected from the rates of L‐NAME hydrolysis. This was explained by accumulation of L‐NOARG in blood cells. 5 These results suggest that L‐NAME represents a prodrug lacking NOS inhibitory activity unless it is hydrolyzed to L‐NOARG. Bioactivation of L‐NAME proceeds at moderate rates in physiological buffers, but is markedly accelerated in tissues such as blood or vascular endothelium.

Attenuation of myocardial ischemia/reperfusion injury in mice with myocyte-specific overexpression of endothelial nitric oxide synthase.

OBJECTIVE The role of nitric oxide (NO) in myocardial ischemia/reperfusion injury remains controversial as both NO donors and NO synthase (NOS) inhibitors have shown to be protective. We generated transgenic (TG) mice that overexpress endothelial NOS (eNOS) exclusively in cardiac myocytes to determine the effects of high cardiac NO levels on ischemia/reperfusion injury and cellular Ca(2+) homeostasis. Wild-type (WT) mice served as controls. METHODS Hearts were perfused in vitro and subjected to 20 min of total no-flow ischemia and 30 min of reperfusion (n=5 per group). Left ventricular function, cGMP levels and intracellular Ca(2+) transients (Ca(2+)(i)) were determined. RESULTS Left ventricular pressure was reduced (maximum, -33%) and basal cardiac cGMP was increased (twofold) in TG hearts, and the changes were reversed by NOS blockade with N(G)-nitro-L-arginine methyl ester (L-NAME). Relative to baseline, recovery of reperfusion contractile function was significantly better in hearts from TG (98%) than WT (51%) mice, and L-NAME abolished this effect. Heart rate and coronary perfusion pressure were not different between groups. Systolic and diastolic Ca(2+)(i) concentrations were similar in WT and TG hearts, but Ca(2+)(i) overload during early reperfusion tended to be less in TG hearts. Kinetic analysis of pressure curves and Ca(2+)(i) transients revealed a faster left ventricular diastolic relaxation and abbreviated aequorin light signals in TG hearts at baseline and during reperfusion. CONCLUSIONS High levels of NO/cGMP strongly protect against ischemia/reperfusion injury, the protection is largely independent of changes in Ca(2+)(i) modulation, but relates to reduced preischemic performance. Myocyte-specific NO augmentation may aid in studies of the (patho)physiological roles of cardiac-derived NO.

Endothelin release during ischaemia and reperfusion of isolated perfused rat hearts

The hypothesis tested was that release of endogenous endothelin plays a role in events associated with or leading to myocardial ischaemia and/or post-ischaemic reperfusion damage. Release of endogenous endothelin into the coronary perfusate of isolated perfused rat hearts during ischaemia and reperfusion was measured with a sensitive radioimmunoassay using a polyclonal antibody with 100% cross-reactivity for all three endothelin isomers. Basal endothelin release was 0.69 +/- 0.02 pg/min/g wet heart weight (n = 35) and was constant up to 180 min. During low-flow hypoxic ischaemia for 180 min (PO2 approximately 250 mmHg) and in the presence of 1% foetal calf serum, the release rate was reduced to below 10% of controls (P < 0.01) and increased four-fold on reperfusion (P = 0.05). The influence of endothelin on vascular and myocardial reperfusion damage was studied with exogenous endothelin-2. After 1 h of low-flow ischaemia, endothelin-2 increased the coronary perfusion pressure to a similar extent as in non-ischaemic hearts, but with a 30-times higher potency. The threshold dose for the constrictive effect was approximately 100 to 300 pg per heart, about ten times more than was recovered in the coronary effluent upon reperfusion. The influence of endothelin on myocardial reperfusion mechanical function (stunning) was assessed with 100 ng endothelin-2, a dose some 3500-fold higher than the amount released during 30 min reperfusion. This dose, given at the onset of reperfusion, improved post-ischaemic aortic output recovery during the first 20 min of reperfusion, but worsened it thereafter (up to 40 min). These data indicate that, in the isolated perfused rat heart model, (1) endothelin is released in measurable amounts into the coronary circulation, (2) the release is much reduced during ischaemia and increased on early reperfusion following prolonged ischaemia, (3) based on the amounts released and the post-ischaemic sensitization of the coronary vasculature to endothelin, the peptide could contribute to reperfusion vascular damage, and (4) endothelin is unlikely to influence stunning owing to the extremely high dose necessary to alter reperfusion mechanical function.

Contribution of aldehyde dehydrogenase to mitochondrial bioactivation of nitroglycerin: evidence for the activation of purified soluble guanylate cyclase through direct formation of nitric oxide

Vascular relaxation to GTN (nitroglycerin) and other antianginal nitrovasodilators requires bioactivation of the drugs to NO or a related activator of sGC (soluble guanylate cyclase). Conversion of GTN into 1,2-GDN (1,2-glycerol dinitrate) and nitrite by mitochondrial ALDH2 (aldehyde dehydrogenase 2) may be an essential pathway of GTN bioactivation in blood vessels. In the present study, we characterized the profile of GTN biotransformation by purified human liver ALDH2 and rat liver mitochondria, and we used purified sGC as a sensitive detector of GTN bioactivity to examine whether ALDH2-catalysed nitrite formation is linked to sGC activation. In the presence of mitochondria, GTN activated sGC with an EC50 (half-maximally effective concentration) of 3.77+/-0.83 microM. The selective ALDH2 inhibitor, daidzin (0.1 mM), increased the EC50 of GTN to 7.47+/-0.93 microM. Lack of effect of the mitochondrial poisons, rotenone and myxothiazol, suggested that nitrite reduction by components of the respiratory chain is not essential to sGC activation. However, since co-incubation of sGC with purified ALDH2 led to significant stimulation of cGMP formation by GTN that was completely inhibited by 0.1 mM daidzin and NO scavengers, ALDH2 may convert GTN directly into NO or a related species. Studies with rat aortic rings suggested that ALDH2 contributes to GTN bioactivation and showed that maximal relaxation to GTN occurred at cGMP levels that were only 3.4% of the maximal levels obtained with NO. Comparison of sGC activation in the presence of mitochondria with cGMP accumulation in rat aorta revealed a slightly higher potency of GTN to activate sGC in vitro compared with blood vessels. Our results suggest that ALDH2 catalyses the mitochondrial bioactivation of GTN by the formation of a reactive NO-related intermediate that activates sGC. In addition, the previous conflicting notion of the existence of a high-affinity GTN-metabolizing pathway operating in intact blood vessels but not in tissue homogenates is explained.

Novel guanylyl cyclase inhibitor, ODQ reveals role of nitric oxide, but not of cyclic GMP in endothelin-1 secretion

The role of nitric oxide (NO) and guanosine 3′,5′‐cyclic monophosphate (cyclic GMP) in cellular regulation of endothelin‐1 (ET‐1) secretion was investigated in cultured porcine aortic endothelial cells. NO synthase was inhibited with (l‐NNA) and guanylyl cyclase with the novel selective inhibitor, ODQ (1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one) (3 μM). Basal and phorbol ester (PMA)‐stimulated ET‐1 secretion were unaffected by ODQ, but stimulated secretion was increased by l‐NNA. In the presence of the NO donors, spermine/ NO, S‐nitroso‐glutathione (GSNO), and nitroprusside (NP) ET‐1 secretion was reduced, but ODQ had no effect on this inhibition, although it effectively inhibited cyclic GMP production. NO release from donors, measured with a sensitive NO electrode, was greatest for spermine/NO, intermediate for GSNO, minimal for NP and paralleled inhibition of ET‐1 secretion. The data suggest that in cultured endothelial cells, curtailment of ET‐1 secretion is mediated by NO and independent of cyclic GMP.

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1 The functional role of the nitric oxide (NO)/guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2 Rat isolated hearts were perfused at constant pressure with Krebs‐Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min−1 for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3 At baseline, heart rate was 287±12 beats min−1, coronary flow was 12.8±0.6 ml min−1, left ventricular developed pressure (LVDevP) was 105±4 mmHg and left ventricular end‐diastolic pressure 4.6±0.2 mmHg in vehicle‐treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05). 4 In normoxic perfused hearts, 1 μM NG‐nitro‐L‐arginine (L‐NOARG) significantly reduced coronary flow from 13.5±0.2 to 12.1±0.1 ml min−1 (10%) and LVDevP from 97±1 to 92±1 mmHg (5%; P<0.05, n=5). 5 Ischaemic contracture was 46±2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 μM) but reduced to ∼30 mmHg (∼25%) at higher concentrations (100 or 1000 μM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L‐NOARG reduced contracture at 1 μM to 26±3 mmHg (23%), but increased it to 63±4 mmHg (59%) at 1000 μM (n=6). Dobutamine (10 μM) exacerbated ischaemic contracture (81±3 mmHg; n=7) and the cyclic GMP analogue Sp‐8‐(4‐p‐chlorophenylthio)‐3′,5′‐monophosphorothioate (Sp‐8‐pCPT‐cGMPS; 10 μM) blocked this effect (63±1 mmHg; P<0.05 vs dobutamine alone, n=5). 6 At the end of reperfusion, LVDevP was 58±5 mmHg, i.e. 55% of pre‐ischaemic value in control hearts, significantly increased to ∼80% by high concentrations of nitroprusside (100 or 1000 μM) or L‐NOARG at 1 μM, while a high concentration of L‐NOARG (1000 μM) reduced LVDevP to ∼35% (P<0.05 vs control; n=6). 7 Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 μM had no sustained effect, but increased cyclic GMP ∼6 fold at 1000 μM; L‐NOARG (1 or 1000 μM) was without effect (n=6). Nitroprusside (1 or 1000 μM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6). 8 In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.

Role of endothelin, nitric oxide and L-arginine release in ischaemia/reperfusion injury of rat heart

OBJECTIVES We tested the hypothesis that endothelin-1 (ET-1) aggravates ischaemia/reperfusion injury by stimulating cellular L-arginine depletion, which would result in reduced synthesis of nitric oxide (NO) and withdrawal of cardioprotection. METHODS Five groups of rat hearts (n = 5 each) were perfused at 9 ml/min per g for 45 min, subjected to 15 min total global ischaemia and reperfused for 30 min; they received, from 5 min pre-ischaemia to end of reperfusion, either vehicle, L-arginine (1 mmol/l), the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; 200 mumol/l), the inhibitor of NO formation NG-nitro-L-arginine (L-NNA; 200 mumol/l), or the ET receptor antagonist PD 142893 (200 nmol/l). Cardiac function and release of L-arginine, cyclic GMP and lactate dehydrogenase (LDH) into coronary effluent were measured. RESULTS Systolic, diastolic, and coronary reperfusion function were consistently improved by L-arginine, SNAP, or PD 142893, but worsened by L-NNA (P < 0.05 in each case). L-arginine release was transiently increased up to 25-fold on reperfusion (vehicle); release was reduced by SNAP (mean: 68%) and entirely prevented by PD 142893. Despite the increased outflow of L-arginine, formation of cyclic GMP was not reduced, but enhanced in reperfusion (11-fold; vehicle), and SNAP further augmented this release, but L-NNA had no significant effect. Release of LDH was decreased by L-arginine, SNAP, and PD 142893 in reperfusion. Finally, release of ET-1 was inhibited by NO in normoxia as well as throughout reperfusion as evident from the stimulatory effect of L-NNA. CONCLUSION In ischaemia, ET-1 cause cell necrosis and L-arginine outflow without compromising NO synthesis in this model.

ROLE OF ETB RECEPTORS IN LOCAL CLEARANCE OF ENDOTHELIN-1 IN RAT HEART : STUDIES WITH THE ANTAGONISTS PD 155080 AND BQ-788

The effects of two endothelin (ET) receptor antagonists, PD 155080 (ETA selective) and BQ‐788 (ETB selective), on cardiac function and ET‐1 release were studied in isolated rat hearts. In normoxic hearts, infusion of PD 155080 (50 nM‐5 μM) reduced coronary resistance, but had no effect on ET‐1 release. Low concentrations of BQ‐788 (2 and 20 nM) had no effect on coronary resistance; high concentrations (0.2 and 2 μM) increased it ∼ 2‐fold; all concentrations increased ET‐1 release (up to 24‐fold). Similar results were obtained in reperfused hearts. Although concentrations of ET‐1 were higher in interstitial fluid than coronary effluent, levels never exceeded the low pg/ml range. These results indicate that (1) ETA receptors mediate coronary constriction, whereas ETB receptors bind and sequester ET‐1, and (2) ET‐1 displaced by ETB antagonist accesses ETA receptors resulting in coronary constriction.

Tissue-toxic effects of phosphatidylcholine/deoxycholate after subcutaneous injection for fat dissolution in rats and a human volunteer.

BACKGROUND The safety of the lipodissolution procedure for the cosmetic treatment of fat is unknown. OBJECTIVES The objective was to determine the subcutaneous tissue effects of phosphatidylcholine solubilized with deoxycholate (PC/DC) in rats and a human volunteer. METHODS Rats were treated subcutaneously three times with 50, 300, or 600 μL of PC/DC formula on the abdomen in a chronic study (30 days). A human volunteer undergoing elective liposuction was similarly treated. Cell membrane lysis, cell viability, and histologic status were determined on fresh biopsies of subcutaneous fat from the injection sites. RESULTS PC/DC dose-dependently reduced membrane integrity and cell viability. Histologic alterations induced by PC/DC included fibroplasia, bandlike fibrosis in the region of the cutaneous muscle, and partial muscle loss. The highest dose caused widespread fat necrosis, fat cyst formation, and necrotic changes of the walls of small blood vessels. Histologic sections of subcutaneous tissue from the human volunteer showed dose-dependent panniculitis, fat cysts, and vessel necrosis. DC (2.5%), tested for comparison in the rat, exerted membrane and histologic effects similar to those of PC/DC. Solvent controls caused negligible alterations. CONCLUSIONS Injection lipolysis with PC/DC causes tissue fibrosis and necrosis of adipose and vascular tissues in rat and man, making the long-term safety of PC/DC for nonsurgical treatment of subcutaneous fat deposits uncertain.