STUDIES ON THE BIPHASIC RELAXANT CURVE OF GLYCERYLTRINITRATE IN RAT AORTA: ROLE OF GTN METABOLITES

1. The present study has examined the possibility that one or more metabolites of glyceryl trinitrate (GTN) (i.e. glyceryl-1,2- and -1,3-dinitrate and glyceryl-1- and -2-mononitrate) may be responsible for the second phase of the biphasic relaxant curve to GTN in phenylephrine-contracted rings of rat aorta. 2. The IC50 values for the two phases of the GTN curve were 0.1 mumol/L and 12 mumols/L with the initial phase eliciting 60% of the total relaxation response. The curves for glyceryl-1,2- and -1,3-dinitrate were monophasic with IC50 values of 248 mumols/L and 110 mumols/L, respectively. The mononitrate metabolites elicited relaxant effects at concentrations greater than or equal to 1 mmol/L. 3. The induction of tolerance to GTN or pretreatment with oxyhaemoglobin (5 mumol/L) resulted in a monophasic GTN curve with IC50 values of 16 mumol/L and 18 mumol/L respectively suggesting selective abolition of responses to low concentrations of GTN with little effect on responses to high concentrations of GTN. The relaxant effects of the -1,2- and -1,3-dinitrates, like that to GTN, were essentially unaltered by GTN tolerance or oxyhaemoglobin. 4. Thus while the relaxant effects of the dinitrate metabolites possess similar properties to that of the second phase of relaxation to GTN, a role for these metabolites is unlikely since their IC50 values are 9-20-fold greater than that for the second phase of relaxation to GTN. Whether these differences are due to the 8-10-fold lower lipophilicity of the dinitrates as compared with the parent compound requires further study.     

Development of tolerance to glyceryl trinitrate in an isolated human peripheral vein and its relation to cyclic GMP metabolism

Human peripheral vein (v. saphena magna) was exposed in vitro to glyceryl trinitrate (GTN) 0.1 mM for 1 h. The subsequent relaxant effect of GTN (10nM-0.1 mM) on vessels contracted by serotonin (0.25 microM) was significantly reduced on vessels preexposed to GTN as compared to control vessels, indicating a development of partial tolerance as far as the vascular smooth muscle relaxant effect is concerned. The impaired relaxant effect was paralleled by a reduced increment of intracellular cyclic GMP. This is, in turn, probably a consequence of both a diminished guanylate cyclase activity and an elevated phosphodiesterase activity in the GTN-tolerant vessels. The relaxant activity as well as the level of intracellular cyclic GMP were restored in GTN-tolerant vessels by dipyridamole (5 microM), an agent with phosphodiesterase inhibiting properties.     

Dissociation of cysteine and glutathione levels from nitroglycerin-induced relaxation

The present study investigated possible involvement of cysteine (CSH) and reduced glutathione (GSH) as critical cellular sulfhydryls which mediate nitroglycerin (GTN)-induced cyclic GMP accumulation and relaxation in bovine coronary artery (BCA). Tolerance to the relaxant effects of GTN was induced in BCA in vitro by preincubation with 1 mM GTN for 2 h. GTN-tolerant BCA were at least 100-fold less sensitive than non-tolerant BCA to the relaxant effects of GTN. Consistent with a relationship between tolerance to both GTN-induced cyclic GMP accumulation and relaxation, cyclic GMP accumulation induced by 1 microM GTN was markedly reduced in GTN-tolerant BCA when compared with non-tolerant BCA. Incubation with 1 mM CSH for 1 h did not significantly alter GTN-induced cyclic GMP accumulation or relaxation in either GTN-tolerant or non-tolerant BCA. Levels of CSH, GSH and glutathione-disulfide (GSSG) were measured in non-tolerant BCA, GTN-tolerant BCA and GTN-tolerant BCA incubated with 1 mM CSH for 1 h. Levels of CSH and GSH were lower in GTN-tolerant BCA than in non-tolerant BCA, whereas GSSG levels were similar in both. In GTN-tolerant BCA incubated with 1 mM CSH, CSH levels were more than 10-fold above, and GSH levels were similar to corresponding values obtained in non-tolerant BCA. These data indicate that although incubation with CSH did not significantly reverse tolerance to GTN-induced cyclic GMP accumulation and relaxation in BCA, it did effectively raise the level of CSH and GSH in GTN-tolerant BCA, at least to corresponding levels found in non-tolerant BCA. These results indicate that the relaxant effects of GTN in BCA do not correlate with tissue levels of CSH and GSH. The findings do not support the hypothesis that CSH and GSH are the cellular sulfhydryls involved in mediating GTN-induced guanylate cyclase activation, cyclic GMP accumulation and relaxation in intact BCA.     

Contribution of activation of K+ channels to glyceryl trinitrate-induced relaxation of rabbit aorta

• 1.1. Possible contribution of K⁺ channel opening to the relaxation by glyceryl trinitrate (GTN) was examined using isolated rabbit aorta.
• 2.2. While glibenclamide and apamin failed to affect relaxation by GTN, both charybdotoxin (ChTx) and iberiotoxin (IbTx) effectively attenuated GTN-induced relaxation.
• 3.3. The increase in cGMP produced by GTN was not attenuated by ChTx and IbTx.
• 4.4. The inhibitory effect of ChTx on GTN-induced relaxation was not reduced in the presence of zaprinast, indicating that cGMP but not GMP was responsible for activation of the K⁺ channel.
• 5.5. Okadaic acid, a selective inhibitor of protein phosphatase 2A, had no effect on the relaxation by GTN. These results indicate that, though small in degree, activation of a ChTx-sensitive K⁺ channel (large conductance Ca²⁺-activated K⁺ channel) is involved in the GTN-induced relaxation in rabbit aorta.

     

Involvement of protein kinase C in reduced relaxant responses to the NO/cyclic GMP pathway in piglet pulmonary arteries contracted by the thromboxane A2-mimetic U46619

1. Impairment of nitric oxide (NO)/cyclic GMP production and/or increased activities of thromboxane A₂ (TXA₂) and endothelin-1 (ET-1) have been associated with pulmonary hypertension. We have analysed the interactions of noradrenaline (NA), the TXA₂-mimetic U46619 and ET-1 with the relaxation induced via cyclic GMP in isolated piglet intrapulmonary arteries.
2. The contractions induced by NA were augmented by endothelium removal or by methylene blue and pre-contracted rings were fully relaxed by acetylcholine, sodium nitroprusside (SNP), atrial natriuretic peptide and 8-bromo-cyclic GMP. In contrast, U46619- and ET-1 induced contractions were endothelium-independent and only partially relaxed by the latter vasodilators. Whereas the reduced responses to SNP in arteries contracted by U46619 were independent of the U46619-induced tone, a higher concentration of ET-1 (tone higher than that induced by NA) was required to reduce the vasodilator responses to SNP. NA, U46619 and ET-1 had no effect on the SNP-induced increases in cyclic GMP.
3. The reduced relaxant responses to SNP in arteries pre-contracted by U46619 were specific for piglet pulmonary arteries since they were not observed in piglet mesenteric or coronary arteries or in rat pulmonary arteries. Furthermore, there were no differences in the relaxant response to the adenylate cyclase activator forskolin in piglet pulmonary arteries pre-contracted by either NA, U46619 or ET-1.
4. SNP-induced relaxation was inhibited by thapsigargin (but not by inhibition of the membrane Na⁺/K⁺ ATPase nor K⁺ channels) indicating a role for Ca²⁺ sequestration by the Ca²⁺ ATPase in the effects of SNP.
5. The phorbol ester 12-myristate, 13-acetate inhibited the relaxant response to SNP. The inhibitory effect of U46619 on SNP-induced relaxation was abolished by the protein kinase C inhibitor (PKC) staurosporine suggesting that PKC may be a part of the signal transduction mechanism.
6. In summary, piglet pulmonary arteries when activated by a TXA₂-mimetic show abnormally reduced relaxant responses to the NO/cyclicGMP pathway. This effect appears to be mediated by activation of PKC.

     

Relaxation of bovine mesenteric arteries by glyceryl trinitrate and other nitro-compounds: evidence for partly different mechanisms of action

The effect of pertussis toxin (PTX) and the cyclic GMP lowering agent LY83583 on the relaxatory response induced by glyceryl trinitrate (GTN), isosorbide dinitrate (ISDN), isosorbide-5-mononitrate (IS-5-MN) and sodium nitroprusside (SNP) in bovine mesenteric artery (BMA) was investigated. Pretreatment with PTX (100 ng/ml; 2 hr induced a 100-fold right shift of the concentration-effect curve for GTN, while no effect on the relaxatory response elicited by ISDN, IS-5-MN or SNP was seen. The relaxatory effect of all the substances tested was markedly reduced by LY83583 (10 microM). The basal cGMP level as well as the GTN induced increase in cGMP were markedly reduced when BMA was exposed to LY83583. The substance also reduced the activation of soluble guanylate cyclase by SNP. Based on the different sensitivity towards PTX it is suggested that GTN induces vascular smooth muscle relaxation by a partly different mechanism than ISDN, IS-5-MN and SNP. As far as the GTN induced relaxation is concerned the sensitivity towards PTX indicates the involvement of regulatory component, possibly a G-protein. However, cyclic GMP seems to play a crucial role in mediating the relaxatory response of all the substances tested since the cGMP-lowering agent LY83583 markedly inhibited the relaxant response induced by all the vascular relaxant agents investigated.     

Inhibition of vascular smooth muscle relaxation by LY83583

Summary The ability of LY83583 to antagonize vascular smooth muscle relaxation elicited by a number of vasodilators was examined in rings of rat aorta. LY83583 (0.3–10 M) inhibited relaxant responses to acetylcholine, calimycin (A23187), adenosine triphosphate (ATP) and sodium nitroprusside, whereas responses to atriopeptin III an activator of particulate guanylate cyclase, and papaverine were unaffected. For acetylcholine and calimycin the major effect of LY83583 (0.3–10 M) was to reduce the maximal response without appreciably altering the EC₅₀ values whereas for ATP the EC₅₀ values were markedly increased by low concentrations of LY83583 (0.3–1 M) with depression of maximal responses occurring at higher concentrations (10 M) of the antagonist. In contrast LY83583 produced nonparallel rightward shifts of the curve for sodium nitroprusside without altering the maximal response. In addition, LY83583 (10 M) reduced basal levels of cyclic GMP and prevented acetylcholine and sodium nitroprusside-induced elevations of cyclic GMP, in parallel with reductions in the relaxant responses. In the presence of LY83583 (10 M) higher concentrations of sodium nitroprusside restored both the relaxant response and the elevation of cyclic GMP. The results of this study show that LY83583 antagonises only those vasodilators which are thought to act via stimulation of soluble guanylate cyclase. The nonsurmountable inhibition of relaxation to acetylcholine, calimycin and ATP probably reflects a limited maximal capacity of the endothelium to release EDRF in response to these agents. Send offprint requests to E. Malta     

Effects of Dipyridamole on the Glyceryl–trinitrate–induced Inhibition of Coronary Artery Muscle Tone and Platelet Aggregation in Relation to Cyclic Nucleotide Metabolism

Abstract: The effects of glyceryl–trinitrate (GTN) and dipyridamole (DIP) on relaxation of bovine coronary arteries and on inhibition of aggregation of human platelets have been studied in vitro with special reference to the cyclic GMP (cGMP) system. GTN had a dose–dependent relaxant effect on bovine coronary arteries, and at a high concentration (10^–5 M) it had an inhibiting effect on platelet aggregation. The effects were associated with an increase in the cGMP levels of the tissues. DIP (5 × 10^–7 M respectively 5 × 10^–6 M) potentiated the coronary artery relaxation induced by GTN (10^–8 M) and the inhibition of platelet aggregation caused by GTN in the concentrations 10^–7–10^–4 M. The potentiation was associated with higher levels of cGMP than those produced by GTN alone, at least in bovine coronary arteries. However, at a concentration of 10^–4 M, GTN, in combination with DIP, caused a significant fall in the cGMP level compared to GTN alone. GTN and DIP were not found to significantly increase the cAMP levels in the concentrations tested. DIP was shown to inhibit phosphodiesterase (PDE) from both platelets and bovine coronary arteries. This might be one of the possible mechanisms that can explain the above mentioned potentiation. It is suggested that the combination of DIP+GTN may be of some clinical importance since the potentiating effects were seen at concentrations comparable to the therapeutic plasma concentration for the respective drugs     

Role of Na+-K+ ATPase in cyclic GMP-mediated relaxation of canine pulmonary artery smooth muscle cells

1. Sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) plays a role in the regulation of vascular tone, but contribution of this enzyme to nitrovasodilator-induced pulmonary vasodilatation remains uncertain. We thus studied the interaction between guanosine 3′:5′-cyclic monophosphate (cyclic GMP) and Na⁺-K⁺ ATPase in smooth muscle cells isolated from canine pulmonary artery.
2. To assess the contractile properties, changes in smooth muscle cell length were determined microscopically. Application of potassium chloride (KCl) shortened the cell length, an effect which was reduced by sodium nitroprusside and 8-bromo-cyclic GMP in a concentration-dependent manner. Pretreatment of cells with the cyclic GMP-dependent kinase inhibitor KT 5823 (2 μM) abolished the effects of sodium nitroprusside and 8-bromo-cyclic GMP.
3. Ouabain (0.3 μM) did not alter the KCl-induced muscle shortening, but inhibited the relaxant responses to sodium nitroprusside and 8-bromo-cyclic GMP.
4. Incubation of smooth muscle cells with sodium nitroprusside concentration-dependently increased intracellular cyclic GMP levels and ouabain-sensitive ⁸⁶Rb uptake, and these values were significantly correlated. In the presence of KT 5823, sodium nitroprusside increased cyclic GMP levels but did not alter ouabain-sensitive ⁸⁶Rb uptake.
5. These results suggest that there is a link between accumulation of intracellular cyclic GMP and activation of sarcolemmal Na⁺-K⁺ ATPase in pulmonary artery smooth muscle cells and that this link may be involved in the sodium nitroprusside-induced pulmonary vasodilatation.

     

Quercetin and its major metabolites selectively modulate cyclic GMP-dependent relaxations and associated tolerance in pig isolated coronary artery

Background and purpose:

Quercetin is a major flavonoid that contributes to the reduced risk of cardiovascular disease associated with dietary ingestion of fruits and vegetables. We have pharmacologically characterized the effect of quercetin, and its sulphate and glucuronide metabolites, on vasoconstrictor and vasodilator responses in the porcine isolated coronary artery.

Experimental approach:

Segments of the porcine coronary artery were prepared for either isometric tension recording or determination of cyclic GMP content. The effect of quercetin and metabolites on submaximal responses to U46619 was examined in the presence and absence of substance P, bradykinin, forskolin, sodium nitroprusside (SNP) and glyceryl trinitrate (GTN).

Key results:

Quercetin and quercetin 3′-sulphate inhibited endothelin and U46619-induced contractions with greater potency (three- to fivefold) against the former, while quercetin 3-glucoronide was inactive. Quercetin enhanced both the cyclic GMP content of the artery (threefold) and cyclic GMP-dependent relaxations to GTN and SNP (two to threefold), but forskolin-induced relaxations were unaffected. Although the effect of quercetin was qualitatively similar to that noted for UK-114,542, a selective inhibitor of phosphodiesterase 5, it was still evident against SNP-induced relaxations in the presence of 10 nM UK-114,542. Quercetin and quercetin 3′-sulphate significantly reduced the development of GTN-associated ‘tolerance’.

Conclusions and implications:

Quercetin and quercetin 3′-sulphate inhibited receptor-mediated contractions of the porcine isolated coronary artery by an endothelium-independent action. Quercetin selectively enhanced cyclic-GMP-dependent relaxations by a mechanism not involving phosphodiesterase 5 inhibition. In addition, quercetin and quercetin 3′-sulphate opposed GTN-induced tolerance in vitro, which may be beneficial for patients treated for angina pectoris.     

Effects of manganese on cyclic GMP levels in the rat ductus deferens

Summary Mn²⁺ and other divalent cations (Me²⁺) are capable of preventing contractile responses of smooth muscle to various stimuli. The effects of Mn²⁺, of other inhibitory Me²⁺ and of Ca²⁺ on basal and hormone-stimulated cyclic nucleotide levels were studied in the ductus deferens of the rat.In tissue segments that had been preincubated with no Me²⁺ added, Mg²⁺ (2 or 10 mM), Co²⁺, Ni²⁺ and Cd²⁺ (2 mM each) caused 2-fold elevations of the cyclic GMP level within 5 min, whereas Mn²⁺ and Ca²⁺ caused 7-and 5-fold elevations, respectively. Cyclic AMP levels were not significantly changed except for a 10–20% increase by Mn²⁺. Whereas Mn²⁺ caused a continuous rise of the cyclic GMP level over at least 10 min, the stimulatory effect of Ca²⁺ was most pronounced during the first minute. The effects of Ca²⁺ and Mn²⁺ were not additive. When Ca²⁺ was added with or after Mn²⁺ (2 mM each), the stimulatory effect of Mn²⁺ was depressed. Similarly, Mn²⁺ caused only a small increase in the cyclic GMP level of tissue incubated with Ca²⁺.With the hormonal stimuli, acetylcholine and noradrenaline (0.1 mM each), a dissociation of their effects on tissue tone and on the cyclic GMP level occurred. Whereas either stimulus caused about 2-fold elevation of the cyclic GMP in the presence of Mn²⁺ with or without Ca²⁺ present, contractile responses were prevented by Mn²⁺. In contrast, K⁺ at a high, depolarizing concentration, which increased the cyclic GMP level in the presence of Ca²⁺, had no effect on the cyclic nucleotide with Mn²⁺ added instead of Ca²⁺. Whereas hormone-induced elevations of the cyclic GMP level depended on the presence of Me²⁺, the effect of hydroxylamine (0.1 mM) to increase the level of this nucleotide in the absence of Me²⁺ was not augmented by Ca²⁺ or Mn²⁺.The present data indicate that hormone-induced elevations of the cyclic GMP level in the absence of extracellular Ca²⁺ do not promote contraction. The findings also suggest that hormones are capable of stimulating cyclic GMP formation by mechanisms other than hormone-induced increase in cytoplasmic Ca²⁺ concentration.Abbreviations cGMP cyclic - GMP guanosine 3:5-monophosphate - cAMP cyclic AMP, adenosine 3:5-monophosphate - Me²⁺ divalent cation(s) - EDTA ethylenediaminetetraacetic acid This work was supported by the Deutsche Forschungsgemeinschaft. A preliminary report was presented (Schultz and Schultz, 1976). The data have partially been taken from a dissertation submitted by K.D.S. to the University of Heidelberg in partial fulfillment of the requirements for the Ph. D. degree     

Effects of KRN2391, a novel vasodilator acting as a nitrate and a K+ channel opener,on the rabbit lower urinary tract

• 1.1. The relaxant effect of KRN2391, suggested to act both as a nitrate and a K⁺ channel opener, was investigated in the rabbit lower urinary tract and compared with the effects of the NO-donor SIN-1 and the K⁺ channel opener levcromakalim.
• 2.2. KRN2391 10⁻⁴ M was able to relax precontracted urethral preparations by 87 ± 4%. Corresponding values for levcromakalim 10⁻⁴ M and SIN-1 10⁻⁴ M were 58 ± 8% and 103 ± 2%, respectively. The -logEC₅₀ values for KRN2391, SIN-1 and levcromakalim were 6.0 ± 0.1, 4.9 ± 0.2 and 5.8 ± 0.2. The relaxant effect of KRN2391 on the bladder was small (29 ± 3%).
• 3.3. The levels of cyclic GMP in the urethral preparations were significantly increased after administration of KRN2391 10⁻⁴ M and SIN-1 10⁻⁴ M, but not after levcromakalim 10⁻⁴ M, the levels measured being 9.9 ± 2.2, 20.9 ± 5.1, and 5.2 ± 1.0, compared to the control value, 3.7 ± 0.5 pmol/mg protein. The levels of cyclic AMP were, however, not changed.
• 4.4. The relaxations, caused by KRN2391 in the urethral preparations, were accompanied by a hyperpolarization (14 ± 4 mV) of the membrane potential.
• 5.5. Methylene blue 3 × 10⁻⁵ M and glibenclamide 10⁻⁵ M significantly reduced the relaxant effect of KRN2391 in the urethral smooth muscle.
• 6.6. We suggest that in the rabbit lower urinary tract, KRN2391 acts mainly as an NO-donor.

     

Effects of endothelin-1 on the isolated guinea-pig ileum: role of Na+ ions

Summary Endothelin-1 induced an unusual transient biphasic effect (relaxation followed by contraction) in the isolated guinea-pig ileum. The contractile component of the response was concentration-dependent in the dose range studied, but the relaxant component was not. Neuronal mechanisms and cyclic GMP do not seem to be involved in the relaxing effect since this component was not affected by either tetrodotoxin, atropine or methylene blue. Endothelin-1 induced desensitization which was well characterized for the contractile component of the response, and was only partially reversed after a 3-h resting period. The effects of endothelin-1 were particularly sensitive to decreases in the Na⁺ gradient induced either by lowering the Na⁺ concentration in the medium or by treating the tissue with ouabain or by a previous treatment with acetylcholine. Amiloride partially inhibited the response induced by endothelin-1 indicating that Na⁺ channels and/or Na⁺/H⁺ exchange are probably involved in its action. Our results suggest that the contractile response induced by endothelin-1 in the isolated guinea-pig ileum depends greatly on Na⁺ ions, in contrast to the responses observed in vascular smooth muscle. Send offprint requests to N. Miasiro at the above address     

Development of tolerance towards glyceryl trinitrate in vitro. Low concentration induced tolerance and changes in cyclic guanosine-3',5'-monophosphate metabolism

Bovine mesenteric arteries were exposed to glyceryl trinitrate (GTN) in vitro for 2 and 8 h in a low concentration (0.1 nmol/l), that equals the plasma concentrations reached by using GTN-discs in patients with angina pectoris. After reexposure to GTN a significantly reduced relaxant response was observed. In the concentration range 1 pmol/l to 10 nmol/l a 10-fold right shift of the concentration-effect curve was noted as compared to control vessels not previously exposed to GTN. The reduced relaxant response was accompanied by a significantly impaired ability to form cyclic guanosine-3',5'-monophosphate (cGMP). The results are interpreted as a development of tolerance towards GTN at the level of the vascular smooth muscle cell. A concentration of GTN (0.1 mmol/l) previously used to induce tolerance in vitro, produced a much more pronounced shift to the right of the concentration-response curve, especially in the concentration range 10 nmol/l to 0.1 mmol/l. This might indicate that tolerance to GTN can be induced by 2 different mechanisms, which in turn might be interpreted as the existence of 2 mechanisms of action by GTN in inducing vascular smooth muscle relaxation.     

Stimulatory effects of brain natriuretic peptide on cyclic GMP accumulation and tyrosine hydroxylase activity in cultured bovine adrenal medullary cells

Summary We studied the effect of brain natriuretic peptide (BNP) on the accumulation of cyclic GMP and the phosphorylation and activity of tyrosine hydroxylase, compared with that of atrial natriuretic peptide (ANP), in cultured bovine adrenal medullary cells. 1. BNP as well as ANP increased cellular cyclic GMP accumulation in a concentration-dependent manner (10–1000 nmol/1). BNP (1 mol/1) and ANP (1 mol/1) produced a 60-fold and 30-fold increase in cyclic GMP accumulation, respectively. 2. The stimulatory effects of BNP and ANP on cyclic GMP accumulation were observed even when Ca²⁺ or Na⁺ was removed from the incubation medium. 3. 12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, inhibited the stimulatory effect of BNP on cyclic GMP accumulation in a concentration-dependent manner (1–100 nmol/1). Furthermore, the BNP-induced accumulation of cyclic GMP was attenuated by forskolin (1 mol/1), an activator of adenylate cyclase. 4. BNP (1 mol/1) and ANP (1 mol/1) caused a significant increase in phosphorylation and activity of tyrosine hydroxylase in the cells. 5. In digitonin-permeabilized cells, cyclic GMP (1–100 mol/1) activated tyrosine hydroxylase in the presence of ATP and Mg²⁺.These results suggest that BNP stimulates the accumulation of cyclic GMP in a manner similar to that of ANP. The increased accumulation of cyclic GMP by these peptides may be negatively modulated by protein kinase C and cyclic AMP and may cause the phosphorylation and activation of tyrosine hydroxylase-in cultured bovine adrenal medullary cells.     

Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP).
2. Using 10 μM of each donor compound, mean±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min⁻¹ (for SNP) to 3.15±0.29 nmol min⁻¹ (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 10⁸ platelets (for SNP) to 2.67±0.31 pmol per 10⁸ platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC).
3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34).
4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8-bromo cyclic GMP).
5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu⁺) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO.
6. Cyclic GMP accumulation in response to GSNO (10⁻⁹–10⁻⁵M) was undetectable following treatment of platelets with ODQ (100 μM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu⁺ ions were required for the cyclic GMP-independent pathway to operate.
7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP.
8. The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (CMPS) produced a concentration-dependent inhibition of aggregation of ODQ-treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP-independent inhibition of platelet aggregation.
9. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP-dependent and -independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP-independent pathway may depend upon a cellular system which involves both copper (I) (Cu⁺) ions and surface membrane thiol groups. The potent anti-platelet action of GSNO results from its ability to exploit this cyclic GMP-independent mechanism.

     

Arginase II inhibition prevents nitrate tolerance

BACKGROUND AND PURPOSE

Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance.

EXPERIMENTAL APPROACH

Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII –/–) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence.

KEY RESULTS

Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration–response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII –/– mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII –/– mice did not.

CONCLUSION AND IMPLICATIONS

Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.     

The Nitric Oxide Donor SIN-1 Is Free of Tolerance and Maintains Its Cyclic GMP Stimulatory Potency in Nitrate-Tolerant LLC-PK1 Cells

Purpose. Using an established cell culture model, the present study investigates whether linsidomine (SIN-1), a spontaneous donor of nitric oxide and active metabolite of the antianginal drug molsidomine, induces tolerance to its own cyclic GMP stimulatory action or shows a diminished response after tolerance induction with glyceryl trinitrate. Methods. Incubations with nitric oxide donors were carried out in LLC-PK₁, kidney epithelial cells. Intracellular levels of cyclic GMP, the vasodilatory second messenger of nitric oxide, were determined by radioimmunoassay. Results. A 5-h preincubation with glyceryl trinitrate (0.01–100 M) led to complete inhibition of a subsequent cyclic GMP stimulation by glyceryl trinitrate but left the cyclic GMP response to SIN-1 unaltered. Similarly, cyclic GMP elevations by the spontaneous nitric oxide donors sodium nitroprusside and spermine NONOate were not affected after pretreatment with glyceryl trinitrate. Moreover, pretreatment with SIN-1 (1–1000 M) had no significant effect on SIN-1-dependent cyclic GMP stimulation. Conclusions. Our results show that in LLC-PK₁, cells, SIN-1 is free of tolerance induction and not cross-tolerant to glyceryl trinitrate. This may be due to the spontaneous nitric oxide release from SIN-1, which in contrast to nitric acid esters does not require enzymatic bioactivation and may therefore be unaffected by nitrate tolerance.     

Different effects of ascorbate deprivation and classical vascular nitrate tolerance on aldehyde dehydrogenase-catalysed bioactivation of nitroglycerin

Background and purpose:

Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance.

Experimental approach:

Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg·kg⁻¹ GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2-and 1,3-glyceryl dinitrate (GDN).

Key results:

Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN ∼10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the ∼10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance.

Conclusions and implications:

These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.     

The effect of chronic treatment with trandolapril on cyclic AMP- and cyclic GMP-dependent relaxations in aortic segments of rats with chronic heart failure

1. Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation.
2. Cardiac output indices of coronary artery-ligated and sham-operated rats were 125±8 and 189±10 ml min⁻¹ kg⁻¹, respectively (P<0.05), indicating the development of CHF at this period.
3. The maximal relaxant response of aortic segments to 10 μM acetylcholine in rats with CHF and sham-operated rats was 64.0±5.7 and 86.9±1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats.
4. In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 μM isoprenaline (44.5±6.7%) was lower that sham-operated rats (81.3±2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by N^G-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 μM dibutyryl cyclic AMP in rats with CHF (26.8±2.7%) was lower than that in sham-operated rats (63.4±11.8%, P<0.05).
5. Trandolapril (3 mg kg⁻¹ day⁻¹) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5±3.6 ml min⁻¹) was lower than that of sham-operated rats (55.0±3.0 ml min⁻¹), and this reduction was reversed (54.1±3.4 ml min⁻¹) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7±1.0%; that to isoprenaline, 80.5±2.8%; that to dibutyryl cyclic AMP, 54.7±6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged.
6. The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.