Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation

This study examined the influence of lowered arterial levels of L-arginine on endothelium-dependent relaxation of isolated rings of bovine pulmonary artery. Incubation of arterial rings under tension for 24 hr in oxygenated Krebs bicarbonate solution at 37 degrees C resulted in the development of marked or complete tolerance to A23187 (calcium ionophore)- and acetylcholine-elicited relaxation. Relaxant responses to nitric oxide were unaffected. Addition of L-arginine did not relax control rings but did elicit marked endothelium-dependent relaxation of tolerant rings that was inhibited by oxyhemoglobin or methylene blue. L-Arginine also restored acetylcholine-elicited relaxation. Inclusion of L-canavanine in the 24 hr incubations protected against the development of tolerance. The tissue concentration of arginine was 3-fold lower in tolerant than control arterial rings and L-canavanine restored arterial arginine levels to control values. Therefore, depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation.     

L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation

The formation of nitric oxide (NO) from L-arginine by vascular endothelial cells and its relationship to endothelium-dependent relaxation of vascular rings was studied. The release of NO, measured by bioassay or chemiluminescence, from porcine aortic endothelial cells stimulated with bradykinin was enhanced by infusions of L-, but not D-arginine. The release of 15NO, determined by high resolution mass spectrometry, from L-guanidino 15N (99%) arginine was also observed, indicating that NO is formed from the terminal guanidino nitrogen atom(s) of L-arginine. L-NG-monomethyl arginine (L-NMMA), but not D-NMMA, inhibited both the generation of NO by endothelial cells in culture and the endothelium-dependent relaxation of rabbit aortic rings. Both these effects were reversed by L-arginine. These data indicate that L-arginine is the physiological precursor for the formation of NO which mediates endothelium-dependent relaxation.     

NG-amino-L-arginine: a new potent antagonist of L-arginine-mediated endothelium-dependent relaxation

This study examined the influence of NG-amino-L-arginine, a novel structural analog of L-arginine, on endothelium-dependent relaxation, contraction, and cyclic GMP accumulation in isolated rings of bovine pulmonary artery. NG-Amino-L-arginine caused potent and stereoselective endothelium-dependent contraction that was associated with a marked and endothelium-dependent decline in basal levels of cyclic GMP in smooth muscle. NG-Amino-L-arginine caused concentration-dependent, competitive, and stereoselective antagonism of acetylcholine-elicited relaxation and cyclic GMP accumulation. NG-Amino-L-arginine was 100- to 300- fold more potent than NG-methyl-L-arginine and did not inhibit endothelium-independent relaxation elicited by nitroglycerin. This potent inhibitory analog of L-arginine should be a useful chemical probe for studying the biosynthesis and biological role of L-arginine-derived nitric oxide both in vitro and in vivo.     

EDHF mediates the relaxation of stretched canine femoral arteries to acetylcholine.

To test the hypothesis that mechanically stretched arteries relax to endothelium-derived vasodilators, we challenged endothelium-intact dog femoral artery rings stretched from 1 to 16 g total initial tension (active force and passive elastic) with 10(-6) M acetylcholine (ACh), an endothelium-dependent dilator. The relaxation to 10(-6) M sodium nitroprusside (SNP), an endothelium-independent dilator, increased with the total initial tension. The relaxation to ACh averaged approximately 65% of the relaxation to SNP at total initial tensions of 4 to 16 g. To determine the nature of the endothelial-derived products involved, we compared the ACh-induced relaxation of stretched rings (6.5 +/- 0.2 g total initial tension) with rings chemically contracted with phenylephrine (Phe, 10(-7) to 10(-5) M) (6.5 +/- 0.3 g total initial tension). ACh-induced relaxation was evaluated before and after the inhibition of the synthesis of eicosanoids [cyclooxygenase (10(-5) M indomethacin) and lipoxygenase (10(-5) M nordihydroguariaretic acid)] and nitric oxide [nitric oxide synthase (10(-5) M Nw-nitro-L-arginine)]. The contribution of endothelium-derived hyperpolarizing factor (EDHF) was identified by blocking calcium-activated potassium channels (10(-8) M iberiotoxin). SNP (10(-6) M) relaxed stretched rings by 1.7 +/- 0.1 g and chemically-activated rings by 4.8 +/- 0.2 g. ACh relaxed stretched rings to 73 +/- 3% of the SNP relaxation and this was only attenuated in the presence of iberiotoxin. ACh relaxed Phe-activated rings to 60 +/- 3% of the SNP relaxation. This relaxation was attenuated by inhibition of the synthesis of nitric oxide and (or) eicosanoids. Therefore, ACh relaxed stretched rings through the release of EDHF whereas the relaxation of chemically activated rings to ACh involved multiple endothelium-derived vasodilators.     

Vasodilatory properties of mono-L-arginine-containing compounds

Benzoyl derivatives of L-arginine, unlike arginine, elicited relaxation of pre-contracted rat aortic rings in a concentration dependent manner. The most potent relaxing agent was N-alpha-benzoyl-L-arginine ethyl ester. The relaxation was abolished by methylene blue, but not by indomethacin. When incubated with rat aortic rings, the benzoyl derivatives exhibited colorimetric reactions characteristic of citrulline and nitrite ion. This indicates the presence of a peptidyl arginine deiminase like activity in rat aorta. Citrulline had no vasodilatory property. The other product of the iminase reaction is ammonia which through oxygenase pathway may generate nitric oxide, the proposed endothelium derived relaxing factor(EDRF). Our results suggest that an as yet unidentified arginine derivative from the endothelium may be the biological precursor of EDRF.     

Possible role of nitric oxide and arachidonic acid pathways in hypoxia-induced contraction of rabbit coronary artery rings

In isolated coronary arteries, hypoxia induces an increase in tone by releasing an unidentified endothelium-derived contracting factor (EDCF). Isometric force was measured in an isolated rabbit coronary artery ring at 37 degrees C in control and high K+ (40 mM) pre-contracted conditions. Hypoxia (15 mmHg pO2) induced by equilibrating the perfusate with nitrogen. Hypoxia did not affect the resting tone but induced an endothelium-dependent contraction on pre-contracted rings. Inhibitors of nitric oxide (NO) were tested, L-NAME (10(-4) M) totally and L-NMMA (10(-4) M) partially convert the hypoxic contraction to an hypoxic relaxation. The addition of L-arginine (10(-4) or 10(-3) M) did not restore the response. Methylene blue (10( -5) M) and ODQ (1 H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one, 10(-5) M), both inhibitors of guanylate cyclase, also changed the hypoxic contraction into a hypoxic relaxation. Catalase (1200 U/ml), which decomposes hydrogen peroxide (H2O2), and superoxide dismutase (150 U/ml, SOD), a free radical scavenger, did not change the hypoxic response but quinacrine (50 microM), an inhibitor of phospholipase A2, significantly decreased it. Inhibitors of arachidonic acid metabolism (indomethacin, diethylcarbamazine, miconazole) however did not affect the hypoxic response. We conclude that in K+ pre-contracted rabbit coronary artery rings, hypoxia induces a contraction which is nitric oxide and arachidonic acid dependent.     

Homocysteine induces endothelial dysfunction via inhibition of arginine transport.

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. High levels of plasma homocysteine (HCY) increase oxidative stress and reduce endothelial-dependent relaxation. We determined whether hyperhomocysteinemia-induced endothelial dysfunction is mediated through inhibition of cellular transport of L-arginine. In endothelial cells, HCY had a biphasic effect on arginine transport. HCY treatment for 6 hr increased L-arginine uptake by 34%; however, uptake was decreased by 25% after 24 h. HCY caused membrane hyperpolarization during both 6 and 24 h incubation periods, indicating that the negative charge facilitating arginine uptake was maintained. HCY significantly reduced expression of cellular arginine transporter protein (CAT-1) after 24 h treatment; whereas endothelial nitric oxide synthase (eNOS) protein levels and basal eNOS activity were not altered. Nevertheless, nitric oxide (NO) formation was significantly decreased. The antioxidant ascorbic acid prevented the effect of HCY on arginine transport. HCY induced formation of the peroxynitrite biomarker nitrotyrosine, which was blocked by supplemental L-arginine. HCY treatment of aortic rings caused decreased vasorelaxation to acetylcholine, which was prevented by supplemental arginine. In conclusion, HCY decreased NO formation and induced endothelial dysfunction without altering protein level or basal activity of eNOS, but through decreases in function and protein expression of the CAT-1 transporter. Reduced arginine supply may lead to eNOS uncoupling and generation of superoxide, contributing to HCY-induced oxidative stress.     

Effect of alpha-lipoic acid on vascular responses and nociception in diabetic rats.

Oxidative stress contributes to the vascular and neurological complications of diabetes mellitus. The aim was to evaluate the effects of treatment with the radical scavenger and transition metal chelator, alpha-lipoic acid, on endothelium-dependent relaxation of the mesenteric vasculature and on superior cervical ganglion blood flow in 8 week streptozotocin-induced diabetic rats. alpha-Lipoic acid effects on small nerve fiber-mediated nociception were also monitored. For the in vitro phenylephrine-precontracted mesenteric vascular bed, diabetes caused a 31% deficit in maximum endothelium-dependent relaxation to acetylcholine, and a 4-fold reduction in sensitivity. alpha-Lipoic acid gave 85% protection against these defects. Acetylcholine responses are mediated by nitric oxide and endothelium-derived hyperpolarizing factor: isolation of the latter by nitric oxide synthase blockade revealed a 74% diabetic deficit that was halved by alpha-lipoic acid. Superior cervical ganglion blood flow, 52% reduced by diabetes, was dose-dependently restored by alpha-lipoic acid (ED(50), 44 mg/kg/d). Diabetic rats exhibited mechanical and thermal hyperalgesia, which were abolished by alpha-lipoic acid treatment. Thus, diabetes impairs nitric oxide and endothelium-derived hyperpolarizing factor-mediated vasodilation. This contributes to reduced neural perfusion, and may be responsible for altered nociceptive function. The effect of alpha-lipoic acid strongly implicates oxidative stress in these events and suggests a potential therapeutic approach.     

Vascular activity of polycations and basic amino acids: L-arginine does not specifically elicit endothelium-dependent relaxation

Irrespective of their stereochemistry (D- or L-form), polycations such as poly-lysine, poly-arginine and poly-histidine elicited endothelium dependent relaxation of pre-contracted rat aortic rings in a dose-dependent manner (ED50 less than or equal to 10-7 M). In contrast, the basic amino acids arginine, glutamine, histidine and lysine caused only endothelium-potentiated relaxation at high concentrations (ED 50 greater than 10-3 M). Both heparin (1U/ml) and dextran sulphate (10 microgram/ml) abolished relaxation by the polycations but had no effect on the responses to the basic amino acids or acetylcholine. These results indicate that the vasodilatory property of the polycations is due to an electrostatic interaction with anionic domains on the endothelial surface, whereas the basic amino acids elicit a non-specific relaxation. Therefore, L-arginine per se cannot be the immediate precursor of nitric oxide, the proposed endothelium-derived relaxing factor.     

Endothelium-dependent relaxation by tetraoctylammonium ions in rat isolated aortic rings

Quaternary ammonium ions are common pharmacological blockers of K+ channels. This study examined the vasorelaxant effect of tetraoctylammonium ions (TOA+) in rat isolated aortic rings. TOA+ caused a concentration-dependent transient relaxation of endothelium-intact tissues. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 3x10(-5) M) or methylene blue (3 x 10(-6) M) or removal of the endothelium abolished the TOA+-induced relaxation. L-arginine (10(-3) M ) partially antagonized the effect of L-NAME. Glibenclamide (3x10(-6) M), charybdotoxin (CTX, 10(-7) M), indomethacin (10(-5) M), or atropine (3x10(-6) M) had no effect. Both TOA+ (10(-5) M)- and acetylcholine (ACh, 10(-5) M)-induced increase in tissue content of cyclic GMP was significantly attenuated by NG-nitro-L-arginine (L-NNA, 10(-4) M) and abolished in endothelium-denuded arteries. These results indicate that TOA+ induced endothelium-dependent relaxation which is likely mediated through nitric oxide but not other endothelium-derived factors. This relaxant action seems unique for TOA+ since other quaternary ammonium ions did not cause nitric oxide-dependent relaxation.     

L-精氨酸产生菌的分子育种

L-精氨酸是一种非常重要的半必需氨基酸,其作为生物体生成NO的前体,参与尿素循环,对人和动物均具有重要的生理功能。现阶段生产精氨酸的主流方法是微生物发酵法,因此,如何快速、高效地选育精氨酸高产菌种成为业内关注的焦点。主要对精氨酸产生菌分子育种方法的最新研究进展进行了综述,并就目前存在的问题和发展方向进行了探讨。     

Endothelium-derived relaxing and contracting factors

Endothelium-dependent relaxation of blood vessels is produced by a large number of agents (e.g., acetylcholine, ATP and ADP, substance P, bradykinin, histamine, thrombin, serotonin). With some agents, relaxation may be limited to certain species and/or blood vessels. Relaxation results from release of a very labile non-prostanoid endothelium-derived relaxing factor (EDRF) or factors. EDRF stimulates guanylate cyclase of the vascular smooth muscle, with the resulting increase in cyclic GMP activating relaxation. EDRF is rapidly inactivated by hemoglobin and superoxide. There is strong evidence that EDRF from many blood vessels and from cultured endothelial cells is nitric oxide (NO) and that its precursor is L-arginine. There is evidence for other relaxing factors, including an endothelium-derived hyperpolarizing factor in some vessels. Flow-induced shear stress also stimulates EDRF release. Endothelium-dependent relaxation occurs in resistance vessels as well as in larger arteries, and is generally more pronounced in arteries than veins. EDRF also inhibits platelet aggregation and adhesion to the blood vessel wall. Endothelium-derived contracting factors appear to be responsible for endothelium-dependent contractions produced by arachidonic acid and hypoxia in isolated systemic vessels and by certain agents and by rapid stretch in isolated cerebral vessels. In all such experiments, the endothelium-derived contracting factor appears to be some product or by-product of cyclooxygenase activity. Recently, endothelial cells in culture have been found to synthesize a peptide, endothelin, which is an extremely potent vasoconstrictor. The possible physiological roles and pathophysiological significance of endothelium-derived relaxing and contracting factors are briefly discussed.     

Different role of endothelium/nitric oxide in 17beta-estradiol- and progesterone-induced relaxation in rat arteries

The present study was aimed to examine the different role of endothelium/nitric oxide in relaxation induced by two female sex hormones, 17beta-estradiol and progesterone in rat isolated aortas and mesenteric arteries. The isometric force of each ring was measured with Grass force-displacement transducers in the organ bathes. 17beta-Estradiol induced both endothelium-dependent and -independent relaxation in the rat aortas but only the endothelium-independent relaxation in the rat mesenteric arteries. In contrast. progesterone induced both endothelium-dependent and -independent relaxation in the rat mesenteric arteries but only endothelium-independent relaxation in rat aortas. N(G)-Nitro-L-arginine methyl ester and methylene blue attenuated the relaxant response to 17beta-estradiol in the aortic rings or to progesterone in the mesenteric arteries. Pretreatment with L-arginine antagonized the effect of N(G)-nitro-L-arginine methyl ester on sex hormone-induced relaxation. The endothelium contribution to relaxation seems to only relate to lower concentrations of 17beta-estradiol and progesterone. In summary, the present results clearly demonstrate a different role of the functional endothelium in the relaxant response to 17beta-estradiol or progesterone in the conduit vessel (aorta) and the resistance vessels (mesenteric artery). Nitric oxide contributes largely to the endothelium-dependent relaxation induced by 17beta-estradiol in the isolated aortas or by progesterone in the mesenteric arteries.     

Comparison of the effect of simvastatin, spironolactone and L-arginine on endothelial function of aorta in hereditary hypertriglyceridemic rats

Hereditary hypertriglyceridemic (hHTG) rats are characterized by increased blood pressure and impaired endothelium-dependent relaxation of conduit arteries. The aim of this study was to investigate the effect of long-term (4 weeks) treatment of hHTG rats with three drugs which, according to their mechanism of action, may be able to modify the endothelial function: simvastatin (an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase), spironolactone (an antagonist of aldosterone receptors) and L-arginine (a precursor of nitric oxide formation). At the end of fourth week the systolic blood pressure in the control hHTG group was 148+/-2 mm Hg and in control normotensive Wistar group 117+/-3 mm Hg. L-arginine failed to reduce blood pressure, but simvastatin (118+/-1 mm Hg) and spironolactone (124+/-4 mm Hg) treatment significantly decreased the systolic blood pressure. In isolated phenylephrine-precontracted aortic rings from hHTG rats endothelium-dependent relaxation was diminished as compared to control Wistar rats. Of the three drugs used, only simvastatin improved acetylcholine-induced relaxation of the aorta. We conclude that both simvastatin and spironolactone reduced blood pressure but only simvastatin significantly improved endothelial dysfunction of aorta. Prominent increase in the expression of eNOS in large conduit arteries may be the pathophysiological mechanism underlying the protective effect of simvastatin in hHTG rats.     

NG-nitro-L-arginine inhibits non-adrenergic, non-cholinergic relaxation in rabbit urethral smooth muscle.

Electrical field stimulation induced a relaxation response in female rabbit urethral smooth muscle strips precontracted with phenylephrine. The relaxation response was inhibited by tetrodotoxin, but not by atropine, propranolol, or hexamethonium. The relaxation response thus results from stimulation of inhibitory non-adrenergic, non-cholinergic nerves. The electrically induced relaxation response was inhibited by an inhibitor of nitric oxide biosynthesis, NG-nitro-L-arginine. This inhibition was overcome by addition of a precursor of nitric oxide, L-arginine. An inhibitor of soluble guanylate cyclase, methylene blue, reduced the relaxation response, and a selective cyclic GMP phosphodiesterase inhibitor, M & B 22948, potentiated the relaxation response. These data indicate that agents which affect the biosynthesis of nitric oxide are associated with the urethral relaxation response evoked by electrical field stimulation, and that cyclic GMP may mediate the relaxation response.     

Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor

Inhibition of the synthesis of endothelium derived relaxing factor by NG-monomethyl-L-arginine, a competitive inhibitor of the synthesis of nitric oxide from L-arginine, enhances hypoxic pulmonary vasoconstriction in pulmonary artery rings and isolated, Krebs albumin perfused rat lungs. L-arginine rapidly reduces hypoxic vasoconstriction, particularly in lungs treated with NG-monomethyl-L-arginine. Following administration of NG-monomethyl-L-arginine, bradykinin-induced vasodilatation is inhibited (p less than 0.01) and a bradykinin-induced vasoconstriction develops (p less than 0.001). NG-monomethyl-L-arginine does not significantly diminish acetylcholine-induced vasodilatation in the isolated lung. NG-monomethyl-L-arginine causes an endothelium-dependent vasoconstriction in pulmonary artery rings.     

11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.     

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

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

双环醇减轻超氧阴离子诱导的大鼠胸主动脉舒张功能损伤

目的：探讨双环醇（bicyclol）对超氧阴离子（O2）诱导的血管舒张功能损伤的影响。方法：采用离体器官灌流技术,观察bicyclol对离体大鼠胸主动脉环张力的影响。采用焦酚（O2的供体）建立O2损伤模型,观察bicyclol预孵育对氧化应激损伤后血管内皮依赖性舒张功能的改善作用。结果：bicyclol（10-8~10-5mol/L）对由苯肾上腺素预收缩的内皮完整主动脉环产生舒张作用,该作用可被NO合酶抑制剂L-NAME和环氧化酶抑制剂吲哚美辛阻断。500μmol/L焦酚可引起乙酰胆碱诱导的主动脉环内皮依赖性舒张反应减弱,bicyclol（10-5mol/L）预孵育45 min可减轻焦酚的损伤作用。对于吲哚美辛处理的主动脉环,bicyclol（10-5mol/L）可抑制焦酚所致的血管舒张反应降低,但这一效应未见于L-NAME处理的主动脉环。结论：bicyclol具有内皮依赖性舒血管作用,并能对抗O2引起的血管舒张功能损伤,该作用通过NO途径介导。     

Does TAME induced contraction involve an endothelium dependent nitric oxide-cyclic GMP mediated pathway?

Two enzyme inhibitors namely L-NAME, a nitric oxide synthase (NOS) inhibitor and methylene blue, a guanylate cyclase inhibitor, were used to elucidate whether N-alpha-tosyl L-arginine methyl ester (TAME)-induced contractions in toad intestinal rings in vitro are mediated through a nitric oxide (NO)- cyclic GMP (c-GMP) pathway. Moreover, a NO precursor, L-arginine was also used to investigate its effect on TAME-induced contractions. Our findings provide evidence that TAME-induced contractions have both an endothelium-dependent and an endothelium-independent component. Based on our findings we now propose that TAME induced contraction involves an endothelium-dependent component mediated through NO and c-GMP.