cardiomyogenesis ͉ cyclic GMP ͉ soluble guanylyl cyclase ͉ NO donors ͉ sGC activators E mbryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo, and because of their selfrenewal and pluripotency, they are thought to revolutionize the field of regenerative medicine (1-3). Although transplantation of stem cells into animal models of cardiac injury and Parkinson's disease has revealed some beneficial effects, a better understanding of the role of specific signaling pathways involved in proliferation and differentiation of ES cells is necessary to improve their use in clinical medicine (4-6). Components of a number of signaling pathways such as Wnt/␤-catenin (7), phosphotidyle-inosital 3-kinase (8, 9), MAPK (10), and NO (11) have been shown to regulate proliferation and differentiation of stem cells.NO is a diffusible short-lived free radical and a signaling molecule with a number of important physiological functions such as smooth muscle relaxation, neurotransmission, and inhibition of platelet aggregation and host defense mechanisms (12). It also plays a role in the pathology of several inflammatory diseases and other pathological conditions such as cancer, diabetes, and neurodegenerative diseases (13,14). NO plays an important role in the control of heart rate, contractibility, coronary perfusion, and cardiac development (15, 16). It is synthesized by enzymes called nitric oxide synthases such as NOS-1 (neuronal NOS), NOS-2 (inducible NOS), and NOS-3 (endothelial NOS) that catalyze the oxidation of L-arginine into L-citruline with the release of NO. NO can also be measured in biological systems as metabolites of NO and as nitrites and nitrates (17). The NO receptor soluble guanylyl cyclase (sGC) is a heme-containing heterodimer with ␣ and ␤ subunits (12). Binding of NO to the heme prosthetic group of sGC catalyzes the conversion of GTP into the second messenger cGMP that can exert many physiological effects, such as mediating vascular smooth muscle tone and motility, phototransduction, and maintenance of fluid and electrolyte homeostasis by interaction of cGMP with downstream effectors such as a family of cGMP-dependent protein kinases, cGMP-dependent phosphodiesterases, and cyclic nucleotide gated channels (11).Our previous studies with mouse and human ES cells demonstrated a time-dependent increase in mRNA and protein levels of different subunits of sGC (with the exception of ␤2 mRNA in H-9 cells) during both mouse and human ES cell differentiation into cells of cardiac lineage. Compared with NOS-1, NOS-2, and NOS-3 mRNA and protein levels were also induced during ES cell differentiation (18,19), thereby demonstrating the involvement of NO signaling components during differentiation of ES cells into cardiac cells. Previous studies by other investigators (20) have shown increased cardiomyogenesis with NO donors and by delivery of the NOS-2 gene in mouse ES cells.In this study, we demonstrate the role of NO and cGMP in differentiation of human and mouse ES cells by regulating t...

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In human erythrocyte membranes incubated with [adenylate‐32P]NAD the 36 kDa protein is predominantly labeled. The labeling is greatly stimulated by nitroprusside in the presence of dithiothreitol. We have purified the 36 k Da protein and identified this modification as crysteine‐specific mono(ADP‐ribosylation) because: (i) labeling occured only when [32P]NAD was replaced by adenine [U‐14C]NAD, but not by [carbonyl‐14C]NAD; (ii) treatment of the prelabeled protein with snake venom phosphodiesterase led to releasing 5′‐[32P]AMP; (iii) the bond between the protein and the nucleotide was hydrolyzed by HgCl2, but was resistant to hydroxylamine. The 36 kDa protein reacted on Western blots with two different monoclonal antibodies (MAbs) against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and was immunoprecipitated by both MAbs.

Here, we review the early studies on cGMP, guanylyl cyclases, and cGMP-dependent protein kinases to facilitate understanding of development of this exciting but complex field of research encompassing pharmacology, biochemistry, physiology, and molecular biology of these important regulatory molecules.

Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO-cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGCα 1 , robust levels of sGC β 1 were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-Amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine); sGC activator), NOC-18+BAY41-2272, IBMX (3-Isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC18+BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo [4,3-a] quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO-sGC-cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms.