A BSTR ACTThe nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H 4 Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O 2 ) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (⅐NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite͞nitrate or the coproduct Cit; we therefore sought to directly quantify ⅐NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable ⅐NO unless superoxide dismutase (SOD; EC 1.15.

The heme-enzyme soluble guanylyl cyclase (sGC) is an ubiquitous NO receptor, which mediates NO downstream signaling by the generation of cGMP. We studied the mechanism of action of the anthranilic acid derivatives 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (HMR1766) (proposed international nonproprietary name, ataciguat sodium) and 2-(4-chloro-phenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)-phenyl)-benzamide (S3448) as a new class of sGC agonists. Both compounds activated different sGC preparations (purified from bovine lung, or crude from human corpus cavernosum) in a concentrationdependent and quickly reversible fashion (EC 50 ϭ 0.5-10 M), with mixed-type activation kinetics. Activation of sGC by these compounds was additive to activation by NO donors, but instead of being inhibited, it was potentiated by the heme-iron oxidants 1H-[1,2,4]-oxdiazolo[3,4-a]quinoxalin-1-one (ODQ) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d) benz(b)(1,4)oxazin-1-one (NS2028), suggesting that the new compounds target the ferric heme sGC isoform. Protoporphyrin IX acted as a competitive activator, and zinc-protoporphyrin IX inhibited activation of hemeoxidized sGC by HMR1766 and S3448, whereas heme depletion of sGC by Tween 20 treatment reduced activation. Both compounds increased cGMP levels in cultured rat aortic smooth muscle cells; induced vasorelaxation of isolated endothelium-denuded rat aorta, porcine coronary arteries, and human corpus cavernosum (EC 50 1 to 10 M); and elicited phosphorylation of the cGMP kinase substrate vasodilator-stimulated phosphoprotein at Ser239. HMR1766 intravenous bolus injection decreased arterial blood pressure in anesthetized pigs. All of these pharmacological responses to the new compounds were enhanced by ODQ and NS2028. Our findings suggest that HMR1766 and S3448 preferentially activate the NO-insensitive heme-oxidized form of sGC, which exists to a variable extent in vascular tissues, and is a pharmacological target for these new vasodilator drugs.The heterodimeric heme-protein soluble guanylyl cyclases (E.C. 4.6.1.2. pyrophosphate lyase, cyclizing; sGC) function as a receptor for the ubiquitous signaling molecule NO. Binding of NO to the ferrous heme activates the enzyme for rapid catalysis of cGMP formation from GTP (Koesling and Friebe, 1999). The second-messenger cGMP triggers several biological processes, such as a decrease in vascular tone and platelet activity, through an interaction with cGMP-specific protein kinases, phosphodiesterases, and ion channels (Munzel et al., 2003). A unifying concept of the molecular requisites for sGC activation has been put forward (Ignarro et al., 1984; Ballou A.M. was supported by grants from Hoechst-Marion-Roussel/Aventis and the German Research Council (SFB 553, project C10).Part of this work was published previously as a thesis (by A.T.) titled Kontrolle der glattmuskulä ren GC-Aktivitä t via NO-Sensitivitä t, Hä m-RedoxStatus und Proteinexpression, 2001 (ISBN 3-930...

1 In this study we examined the endothelium-dependent e ect of YC-1 ± a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC) ± on vascular relaxation and nitric oxide (NO) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. 2 In preconstricted rat aortic rings with intact endothelium, YC-1 produced a concentrationdependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC-1, when (i) the aortas were pre-treated with L-N G -nitroarginine methylester (L-NAME) or (ii) the endothelium was removed. 3 Incubation of bovine aortic endothelial cells (BAEC) with YC-1 produced a concentrationdependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre-incubating cells with L-NAME or with 8-bromo-cyclic GMP decreased this e ect indicating that the YC-1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct e ect of YC-1 on recombinant endothelial constitutive NO synthase activity was observed. 4 The YC-1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. 5 In human umbilical vein endothelial cells (HUVEC), YC-1 stimulated intracellular cyclic GMP production in a concentration-and time-dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC-1 compared to calcium ionophore A23187. Similar e ects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). 6 When HUVEC and RMCEC were pre-treated with L-N G -nitroarginine (L-NOARG), the maximum YC-1 stimulated cyclic GMP increase was reduced by 550%. 7 These results indicate, that beside being a direct activator of sGC, YC-1 stimulates a NOsynthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.

The term "nootropic" refers to compounds that act on cognitive functions. These drugs should facilitate learning and memory and prevent impairment of cognitive functions induced by diseases and brain insults. In animal models of impaired cognitive functions, the effects of several nootropic substances were investigated. Learning and retention in passive avoidance, and positive reinforcement paradigms were disrupted by hypoxia, cerebral ischemia and amnesia-inducing agents. Piracetam and several other novel nootropic compounds were shown to improve performance in these animal models. These results indicate that experimentally induced cognitive dysfunction in animals can be attenuated with drug treatment.