cGMP Accumulation Research Articles

A Candidate Hypertension Gene

See related article, pages 992–999 Hypertension affects >20% of the general population, and yet its etiologic basis remains unknown in the vast proportion of those affected.1 Hypertension greatly increases the risk of stroke, myocardial infarction, congestive heart failure and renal dysfunction thus making it an important focus of clinical research. Although pharmacological reductions in blood pressure have been shown to decrease the incidence of these adverse consequences,2 large numbers of hypertensive patients go undiagnosed, undertreated, or are nonresponsive to lifestyle modifications and medical therapy.1 As such, there remains a pressing need for an improved understanding of the mechanisms underlying hypertension. Recent advances in genomic and proteomic analyses have led to the discovery of Mendelian forms (monogenetic traits) of hypertension.3,4 Although rare, these mutations which mainly involve altered renal salt handling, provide a molecular basis for understanding the critical role of the kidney during normal blood pressure regulation.5 By contrast, our understanding of the relative contributions of kidney, heart, CNS, and blood vessel function to blood pressure variations in the general population is complicated by the fact that spontaneous hypertension typically arises as a complex quantitative trait affected by differing combinations of genetic and environmental factors. A number of quantitative trait loci (QTL) associated with hypertension have been identified in both animal disease models and human patients, however the affected gene(s) at these sites have remained elusive owing to the large size and complexity of the regions identified.6 The use of congenic rodent strains to reduce the genomic size of QTLs is an important advance in the field (Reviewed in 7). Indeed, an article in this issue of Circulation Research highlights the potential of …

Nitroxyl Anion Donor, Angeli’s Salt, Does Not Develop Tolerance in Rat Isolated Aortae

The nitroxyl anion (HNO) is emerging as a novel regulator of cardiovascular function with therapeutic potential in the treatment of diseases such as heart failure. It remains unknown whether tolerance develops to HNO donors, a limitation of currently used nitrovasodilators. The susceptibility of the HNO donor, Angeli's salt (AS), to the development of vascular tolerance was compared with the NO donors, glyceryl trinitrate (GTN) and diethylamine/NONOate (DEA/NO) in rat isolated aortae. Vasorelaxation to AS was attenuated (P<0.01) by the HNO scavenger l-cysteine, whereas the sensitivity to GTN and DEA/NO was decreased (P<0.01) by the NO. scavenger carboxy-[2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidozoline-1-oxy-3-oxide]. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one impaired responses to GTN>or=AS>>DEA/NO. Pretreatment with 10, 30, and 100 micromol/L of GTN for 60 minutes induced a 4- (P<0.05), 13- (P<0.01), and 48-fold (P<0.01) decrease in sensitivity to GTN, demonstrating tolerance development. In contrast, pretreatment with AS or DEA/NO (10, 30, and 100 micromol/L) did not alter their subsequent vasorelaxation. All of the nitrovasodilators (30 micromol/L) displayed a similar time course of vasorelaxation and cGMP accumulation over a 60-minute period. Unlike vasorelaxation, the magnitude of peak cGMP accumulation differed substantially: DEA/NO>>AS>GTN. GTN did not induce cross-tolerance to either AS or DEA/NO. In contrast, pre-exposure to DEA/NO, but not AS, caused a concentration-dependent attenuation (P<0.01) of GTN-mediated relaxation, which was negated by the protein kinase G inhibitor guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,Rp-isomer, triethylammonium salt. In conclusion, vascular tolerance does not develop to HNO, nor does cross-tolerance between HNO and GTN occur. Thus, HNO donors may have therapeutic advantages over traditional nitrovasodilators.

Small GTPase Rac Links Receptors to cGMP Formation

The second messenger guanosine 3′,5′-monophosphate (cGMP) controls a broad range of cellular functions, many of which are regulated through the cGMP-dependent protein kinase. Cyclic GMP is synthesized by guanylyl cyclases (GCs) that catalyze their formation from guanosine triphosphate. A study by Guo et al . provides insight into the upstream regulation of the GCs. Unlike the adenyl cyclases, which are well known as targets of GPCRs [heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors], transmembrane guanylyl cyclases are thought to act as receptors themselves, although not all have identified ligands. Guo et al . explored how intracellular signals can also regulate the transmembrane guanylyl cyclase GC-E and observed that transient overexpression of the small GTPase Rac1 led to activation of GC-E. Activation of the platelet-derived growth factor receptor (PDGFR) also stimulated formation of cGMP, and this was inhibited when the authors used a dominant-negative form of Rac1 to inhibit activity of endogenous Rac1. The authors tested known targets of Rac1 to explore how Rac1 might be coupled to activation of GC-E. PAK (p21-activated protein kinase) is one such target, and it could substitute for Rac1 in activating formation of cGMP; expression of the autoinhibitory domain of PAK also inhibited the effect of constitutively active Rac1 to stimulate GC-E activity. The authors used in vitro and in vivo studies with purified proteins and protein fragments to show that PAK directly interacts with and stimulates GC-E. This activation required the kinase activity of PAK--but apparently only for autophosphorylation, because GC-E was not phosphorylated by PAK. This new pathway from receptor tyrosine kinase to cGMP was shown to be physiologically important in the control of cell migration. Depletion of GC-A in mouse embryo fibroblasts with small interfering RNA (siRNA) reduced the effect of PDGF on accumulation of cGMP and inhibited PDGF-dependent cell migration and formation of lamellipodia. Settleman discusses the work in a commentary and emphasizes some of the intriguing questions raised by the work, such as why Cdc42, another small GTPase that also activates PAK, fails to activate GC-E. D. Guo, Y.-C. Tan, D. Wang, K. S. Madhusoodanan, Y. Zheng, T. Maack, J. J. Zhang, X.-Y. Huang, A Rac-cGMP signaling pathway. Cell 128 , 341-355 (2007). [Online Journal] J. Settleman, PAK-in’ up cGMP for the move. Cell 128 , 237-238 (2007). [Online Journal]

Mechanisms underlying the chronic pioglitazone treatment-induced improvement in the impaired endothelium-dependent relaxation seen in aortas from diabetic rats

The objectives of this study were to determine the effects of chronic treatment with pioglitazone, a peroxisome proliferator-activated receptor γ agonist, on the impaired endothelium-dependent relaxation seen in aortas from established streptozotocin (STZ)-induced diabetic rats, and to identify some of the molecular mechanisms involved. Starting at 8 weeks of diabetes, pioglitazone (10 mg/kg) was administered to STZ-induced diabetic rats for 4 weeks. In untreated STZ rats (vs age-matched control rats): (1) ACh-induced relaxation, cGMP accumulation, phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein at Ser-239 [an established biochemical end-point of nitric oxide (NO)/cGMP signaling], and Cu/Zn-superoxide dismutase (SOD) expression and SOD activity were all reduced; (2) aortic superoxide generation, nitrotyrosine expression, and NAD(P)H oxidase activity were increased; (3) plasma endothelin-1 (ET-1) and aortic c-Jun (AP-1 component) protein expressions were increased. Pioglitazone treatment markedly corrected the above abnormalities. Collectively, these results suggest that pioglitazone treatment improves endothelium-dependent relaxation by reducing oxidative stress via increased SOD activity, decreased NAD(P)H oxidase activity, and a decreased ET-1 level, and that this decreased ET-1 level may be attributable to an inhibition of the AP-1 signaling pathway.

Mechanisms of Oxidative Stress-Induced Increase in Salt Sensitivity and Development of Hypertension in Sprague-Dawley Rats

High salt intake produces vascular changes that contribute to the development of hypertension in salt-sensitive individuals. Because reactive oxygen species play a role in the pathogenesis of cardiovascular diseases, we investigated whether oxidative stress contributes to salt-sensitive hypertension. Sprague-Dawley rats were divided in different groups and received tap water (vehicle), 30 mmol/L of l-buthionine sulfoximine ([BSO] an oxidant), high salt ([HS] 1% NaCl), and BSO plus HS without and with antioxidant tempol (1 mmol/L) in drinking water for 12 days. Compared with vehicle, BSO treatment caused oxidative stress and mild increase in blood pressure. Thoracic aortic rings from BSO-treated rats exhibited decreased response to endothelium-independent vasorelaxants. In HS-treated rats, the response to vasoactive agents, as well as blood pressure, was unaffected. Concomitant treatment of rats with BSO and HS produced a marked increase in blood pressure and a decreased response to both endothelium-dependent and endothelium-independent vasorelaxants with an increase in EC(50). Incubation of aortic tissue from BSO-treated rats with sodium nitroprusside showed decreased cGMP accumulation, whereas HS rats had decreased basal NO synthase activity. Tempol decreased oxidative stress, normalized blood pressure, and restored NO signaling and responses to vasoactive compounds in BSO and BSO plus HS rats. We conclude that BSO increases oxidative stress and reduces NO signaling, whereas HS reduces NO levels by decreasing the NO synthase activity. These phenomena collectively result in reduced responsiveness to both endothelium -dependent and endothelium- independent vasorelaxants and may contribute to salt-sensitive hypertension.

ACh‐stimulated exocytosis enhanced by arachidonic acid in guinea pig antral mucous cells: cGMP accumulation via PPARα

Arachidonic acid (AA, 10 nM – 2 μM) enhanced Ca2+-regulated exocytosis in acetylcholine (ACh) stimulated antral mucous cells of guinea pig. The enhancement in ACh-stimulated exocytosis induced by AA was not mediated via PGE2 accumulation. The AA actions were inhibited by MK886 (50 μM, an inhibitor of by peroxisome proliferation activation receptor ? (PPAR?), and were mimicked by PPAR? agonists (Eicosatetraynoic acid (ETYA) and WY14643). The enhancement of ACh-stimulated exocytosis induced by AA, ETYA and WQY14643 was eliminated by a PKG inhibitor (Rp8BrPET-cGMPS) and was mimicked by 8Br-cGMP. Nitro-L-arginine methyl ester (L-NAME) also inhibited the enhancement induced by AA, ETYA and WY14643, and 1-Hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-1-triazene (NOC-12) enhanced ACh-stimulated exocytosis similar to AA, ETYA and WY14643, which was inhibited by Rp8BrPET-cGMPS. AA, ETYA or WY14643 did not increase intracellular Ca2+ concentration ([Ca2+]i) and did not enhance ACh-stimulated increases in [Ca2+]i. Measurements of cGMP contents in antral mucosa demonstrated that stimulation with ACh, AA and PPAR? agonists stimulate cGMP accumulation, which is inhibited by MK886 and L-NAME. In conclusion, in ACh-stimulated antral mucous cells, AA accumulated stimulates PPAR?, which activates the NO/cGMP cascade, and PKG activated by cGMP enhances Ca2+-regulated exocytosis in antral mucous cells by increasing number of the primed granule.μ μ μ μ μ

PAK-in' Up cGMP for the Move

Although guanylyl cyclases have been implicated in cell migration, how they are activated to promote cell motility is unknown. In this issue, report direct communication between guanylyl cyclases and the Rac-p21-activated kinase (PAK) signaling pathway--which is essential for cell migration--to promote cell motility, through allosteric activation of guanylyl cyclases by autophosphorylated PAK.

A Rac-cGMP Signaling Pathway

The small GTPase Rac and the second messenger cGMP (guanosine 3',5'-cyclic monophosphate) are critical regulators of diverse cell functions. When activated by extracellular signals via membrane signaling receptors, Rac executes its functions through engaging downstream effectors such as p21-activated kinase (PAK), a serine/threonine protein kinase. However, the molecular mechanism by which membrane signaling receptors regulate cGMP levels is not known. Here we have uncovered a signaling pathway linking Rac to the increase of cellular cGMP. We show that Rac uses PAK to directly activate transmembrane guanylyl cyclases (GCs), leading to increased cellular cGMP levels. This Rac/PAK/GC/cGMP pathway is involved in platelet-derived growth factor-induced fibroblast cell migration and lamellipodium formation. Our findings connect two important regulators of cellular physiological functions and provide a general mechanism for diverse receptors to modulate physiological responses through elevating cellular cGMP levels.

Statins Activate AMP-Activated Protein Kinase In Vitro and In Vivo

Statins exert pleiotropic effects on the cardiovascular system, in part through an increase in nitric oxide (NO) bioavailability. AMP-activated protein kinase (AMPK) plays a central role in controlling energy and metabolism homeostasis in various organs. We therefore studied whether statins can activate AMPK, and if so, whether the activated AMPK regulates nitric oxide (NO) production and angiogenesis mediated by endothelial NO synthase, a substrate of AMPK in vascular endothelial cells. Western blotting of protein extracts from human umbilical vein endothelial cells treated with atorvastatin revealed increased phosphorylation of AMPK at Thr-172 in a time- and dose-dependent manner. The AMPK activity, assessed by SAMS assay, was also increased accordingly. The phosphorylation of acetyl-CoA carboxylase at Ser-79 and of endothelial NO synthase at Ser-1177, 2 putative downstream targets of AMPK, was inhibited by an adenovirus that expressed a dominant-negative mutant of AMPK (Ad-AMPK-DN) and compound C, an AMPK antagonist. The positive effects of atorvastatin, including NO production, cGMP accumulation, and in vitro angiogenesis in Matrigel, were all blocked by Ad-AMPK-DN. Mice given atorvastatin through gastric gavage showed increased AMPK, acetyl-CoA carboxylase, and endothelial NO synthase phosphorylation in mouse aorta and myocardium. Statins can rapidly activate AMPK via increased Thr-172 phosphorylation in vitro and in vivo. Such phosphorylation results in endothelial NO synthase activation, which provides a novel explanation for the pleiotropic effects of statins that benefit the cardiovascular system.

In vitro effects of homologous natriuretic peptides on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus

C-type natriuretic peptide (CNP) cDNA was cloned from the tilapia brain and its inferred mature sequence was chemically synthesized together with previously cloned tilapia A-type and B-type natriuretic peptides (ANP and BNP). The cloned CNP belongs to the CNP-1 type of teleosts. Reverse-transcription polymerase chain reaction showed that the ANP and BNP genes were hardly expressed in the tilapia brain and pituitary, whereas the CNP gene was expressed strongly in the brain and slightly in the pituitary. Effects of homologous natriuretic peptides (100 nM each) on growth hormone (GH) and prolactin (PRL) release were examined using dispersed tilapia pituitary cells. Tilapia ANP and BNP stimulated GH and PRL release during 4–8, and 8–24 h of incubation. BNP appeared to be more potent than ANP, also stimulating GH and PRL release during 0–4 h of incubation. CNP stimulated GH release only during 4–8 h of incubation; CNP was without effect on PRL release. All three NPs stimulated GH and PRL mRNA expression in dispersed pituitary cells following 24 h of incubation. ANP and BNP significantly elevated intracellular cGMP accumulation in dispersed pituitary cells after 15 min of exposure, whereas no effect of CNP was observed. These results indicate a long-lasting stimulation of GH and PRL release by ANP and BNP that is mediated, at least in part, by the guanylyl cyclase-linked NP receptor.

Survey of Literature

Mesenchymal stem cells alone or ex vivo gene modified with endothelial nitric oxide synthase reverse age-associated erectile dysfunction. TJ Bivalacqua, W Deng, M Kendirci, MF Usta, C Robinson, BKTaylor, SNMurthy, HCChampion, WJG Hellstrom, PJ Kadowitz. Am J Physiol 2007; 292:H1278–90. Overexpression of arginase in the aged mouse penis impairs erectile function and decreases eNOS activity: Influence of in vivo gene therapy of anti-arginase. TJ Bivalacqua, AL Burnett, WJG Hellstrom, HC Champion. Am J Physiol 2007;292:H1340–51. Editorial Comment: Investigations into the molecular mechanisms responsible for ageassociated erectile dysfunction in the absence of overt disease have been undertaken only within the past decade. These two studies illustrate different, but effective, approaches in ameliorating erectile dysfunction in aged animals. In the first study, investigators isolated mesenchymal stem cells (MSCs) from bone marrow and injected them directly into the penile corpus cavernosum. Consistent with earlier studies by the same investigators, MSCs initially expressed markers for both smooth muscle (smooth muscle actin) and endothelial cells (von Willebrand factor) and survived within the corpus cavernosum for up to 21 days after injection with no signs of tissue inflammation. After 21 days,MSCs exhibited an alteration in phenotype, expressing additional proteins characteristic of smoothmuscle and endothelial cells that were not present at the time of isolation. Most importantly, aged rats injected with MSCs exhibited increases in endothelial nitric oxide synthase (eNOS) protein, enzyme activity, and cyclic guanosine monophosphate (cGMP) accumulation with enhanced erectile function at 21 days. Intracavernosal injection of modified MSCs, infected with adenovirus containing the eNOS gene, improved erectile function in aged rats as early as 7 days.While further studies are required to determine the feasibility of adult stem cell therapy in humans, these findings provide more evidence for the utility of MSCs in regenerative medicine and highlight the remarkable plasticity of stem cells and their ability to adapt to their local tissue environment. In the second study, investigators characterized changes in arginase and eNOS in aged mice. Erectile function in response to nerve stimulation or endothelium-dependent vasodilator was decreased as a function of age, whereas dietary intake of L-arginine for 2 weeks reversed this trend. Interestingly, mRNA and protein for both arginase I and eNOS were upregulated in endothelial cells isolated from penile tissue of aged mice. However, while arginase enzyme activity was also increased, NOS enzyme activity and baseline cGMP production was lower in penile tissue from aged mice. Because L-arginine serves as a common substrate for arginase and eNOS, the increased arginase activitymay decrease the available substrate pool to inhibit nitric oxide production. Indeed, cGMPproduction was increased to a greater extent in tissue extracts from aged rats when the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) was included. Further, intracavernosal administration of ABH normalized or enhanced erectile responses in aged mice. Intracavernosal injection of adenovirus containing an antisense sequence for the arginase-I gene decreased arginase-I expression and increased erectile function, NOS activity, and cGMP production in aged mice 1 month after infection. While it is likely that multiple factors contribute to the decline in erectile function with age, these data demonstrate that upregulation of arginase I contributes to endothelial dysfunction in penile corpus cavernosum of aged animals. This thorough study enhances our appreciation for perturbations in arginine metabolism that are increasingly being related to different pathological processes in a wide variety of organ systems. Noel N. Kim, PhD

Overexpression of arginase in the aged mouse penis impairs erectile function and decreases eNOS activity: influence of in vivo gene therapy of anti-arginase

Since both increased nitric oxide (NO) synthase (NOS) abundance and diminished NO signaling have been reported in the aging penis, the role of NO in the adaptations of aging remains controversial. Here we tested the hypothesis that arginase, an enzyme that competes with NOS for the substrate l-arginine, contributes to erectile dysfunction with advanced age in the B6/129 mouse strain. Arginase protein abundance, mRNA expression, and enzyme activity were elevated in aged compared with young penile endothelial cells. In addition, endothelial NOS (NOS3) protein abundance was greater in aged versus young penile endothelial cells, whereas NOS activity and cGMP levels were reduced. Calcium-dependent l-arginine-to-l-citrulline conversion and cGMP formation increased significantly in aged mouse penes in the presence of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH). However, there was no effect on l-arginine-to-l-citrulline conversion or cGMP accumulation in the endothelium from young mouse penes. To assess the functional role of arginase in the inhibition of NOS pathway responsiveness in the penis, we evaluated the effects of ABH and an adeno-associated virus encoding an antisense sequence to arginase I (AAVanti-arginase) on erectile function in vivo. ABH and AAVanti-arginase enhanced endothelium-dependent erectile responses in the aged mice without altering endothelium-independent responses. Paralleling our in vitro observations, ABH or AAVanti-arginase did not affect vascular responses in the young mice. Inhibition of the arginase pathway improves endothelial function in the aging penile circulation, suggesting that the arginase pathway may be exploited to improve erectile dysfunction associated with aging.

Cardiovascular Effects of Phosphodiesterase 5 Inhibitors

Phosphodiesterase 5 inhibitors, such as sildenafil, vardenafil and tadalafil, are now approved for the treatment of erectile dysfunction. They inhibit the cGMP-specific isoform 5 of phosphodiesterase, resulting in cGMP accumulation, which, for example in smooth muscle cells, reduces muscular tone. In the cardiovascular system, they slightly reduce arterial systemic blood pressure. This moderate effect was also shown in combination with many antihypertensive drugs. But the important contraindication is the concomitant use of PDE 5 inhibitors with any drug serving as a nitric oxide donor, as this combination can lead to significant arterial hypotension. Caution is needed in patients on alpha-blocking agents. In general, this class of drugs was not shown to exhibit direct deleterious effects on the myocardium or promote arrhythmias. Furthermore, statistical evaluations did not demonstrate an increased risk for patients taking PDE 5 inhibitors in comparison with an adequate control population. Many patients suffering from erectile dysfunction may be characterized by multiple cardiovascular risk factors or even ischemic heart disease, suggesting an increased baseline risk. While in many forms of erectile dysfunction, these agents seem to be very effective, it becomes clear that endothelial dysfunction is an attractive target of PDE 5 inhibitors and may also be the underlying cause in many types of erectile dysfunction. In addition, these agents seem to be very effective in lowering pulmonary arterial pressure, which might provide the opportunity to treat primary and some forms of secondary pulmonary hypertension, perhaps in combination with inhaled nitric oxide or other pulmonary arterial vasodilators. Sildenafil was approved for treatment of primary arterial hypertension in the U.S. in June 2005. Recently, direct cardioprotective effects were described in animal research, resembling preconditioning-like effects, which may, under certain conditions, also be applicable in clinical research.

Effect of icariin on cyclic GMP levels and on the mRNA expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in penile cavernosum

To further investigate the mechanisms of action of icariin (ICA), we assessed the effects of ICA on the in vitro formation of cGMP and cAMP in isolated rabbit corpus cavernosum. Isolated segments of rabbit corpus cavernosum were exposed to increasing concentrations of ICA and the dose-dependent accumulation of cGMP and cAMP was determined in the tissues samples by means of 125I radioimmunoassay. Responses of the isolated tissues preparations to ICA were compared with those obtained with the reference compounds sildenafil (Sild). Furthermore, the effects of ICA on the mRNA expression of specific cGMP-binding phosphodiesterase type V (PDE5) in rat penis were also observed. After incubation with ICA for 6 h or 14 h respectively, the levels of PDE5 mRNA were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that ICA increased cGMP concentrations directly (P < 0.05), but there was no significant effect on cAMP concentrations (P > 0.05). In the presence of sodium nitroprusside (SNP), a stimulatory agent of cGMP, both ICA and Sild increased cGMP concentrations with increasing dose (P < 0.01). Their EC50 was 4.62 (ICA) and 0.42 (Sild) micromol/L respectively. Under the same condition, ICA and Sild unaltered cAMP level significantly (P > 0.05). There were PDE5A1 and PDE5A2 mRNA expressions in rat corpus cavernosum with PDE5A2 being the dominant isoform. ICA could obviously inhibit these two isoforms mRNA expression in rat penis, and decrease PDE5A1 more pronouncedly (P < 0.01). The present study indicated that the aphrodisiac mechanisms of icariin involved the NO-cGMP signal transduction pathway, with increasing cGMP levels in the corpus cavernosum smooth muscle. The inhibitory effect of icariin on PDE5 mRNA expression, especially on PDE5A1, might account for its molecular mechanisms for its long-term activity.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 μM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) (100 μM) or inhibiting PDI with bacitracin (5 mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 μM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 μM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-( N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Endothelin Signaling Pathways in Rat Adrenal Medulla

1. We further characterized the effect of endothelins (ETs) on receptor-mediated phosphoinositide (PI) turnover, nitric oxide synthase (NOS) activation, and cGMP formation in whole rat adrenal medulla. 2. The PI hydrolysis was assessed as accumulation of inositol monophosphates (InsP(1)) in the presence of 10 mM LiCl in whole tissue and the analysis of inositol-1-phosphate by Dowex anion exchange chromatography. NOS activity was assayed by monitoring the conversion of radiolabeled L-arginine to L-citrulline. Cyclic GMP formation was assessed as accumulation of cGMP in whole tissue in the presence of phosphodiesterase inhibition, and the amount of cGMP formed was determined by radioimmuno-antibody procedure. 3. ET-1 and ET-3 increased PI turnover by 30% in whole adrenal medulla prelabeled with [(3)H] myoinositol. Both ETs isoforms, at equimolar doses, increased NOS activity and cGMP levels in similar degree. The selective ET(B) receptor agonist, IRL-1620, also increased cGMP formation, mimicking the effects of ETs, while IRL-1620 did not alter the PI metabolism. ETs-induced InsP(1) accumulation and cGMP was dependent on extracellular calcium. The effect of ETs on PI turnover was inhibited by neomycin. The L-arginine analogue, N-nitro-L-arginine (L-NAME), and two inhibitors of soluble guanylyl cyclase, methylene blue and ODQ, significantly inhibited the increase in cGMP production induced by ETs or IRL-1620. The selective ET(A) receptor antagonist, BQ 123, inhibited the ETs-induced increase in PI turnover, while the selective ET(B) receptor antagonist, BQ 788, was ineffective. Likewise, BQ 788, significantly inhibited ET-1- or ET-3-induced NOS activation and cGMP generation but not ETs-induced InsP(1) accumulation. 4. Our data indicate that stimulation of PI turnover and NO-induced cGMP generation constitutes ETs signaling pathways in rat adrenal medulla. The former action is mediated through activation of ET(A) receptor, while the latter through the activation of ET(B) receptor. These results support the role of endothelins in the regulation of adrenal medulla function.

Nitric oxide-evoked cGMP production in Purkinje cells in rat cerebellum: An immunocytochemical and pharmacological study

The cerebellar cells that account for glutamate-dependent cyclic GMP (cGMP) production, involving activation of the ionotropic glutamate receptors/nitric oxide synthase/soluble guanylyl cyclase pathway, are not fully established. In the present paper we have searched for the localisation of the cGMP response to the nitric oxide (NO) donor S-nitroso-penicillamine (SNAP 1 μM), expected to generate local NO concentrations in the low nanomolar physiological range and evoking a cGMP response dependent on glutamate release and on the consequent activation of ionotropic glutamate NMDA/non-NMDA receptors , in cerebellar slices from adult rat. We have found that low concentration of exogenous NO evoked cGMP accumulation in Purkinje cells in an ionotropic glutamate receptor-dependent and tetrodotoxin-sensitive manner. Such immunocytochemical localisation appears consistent with functional evidence for physiologically relevant glutamate-dependent cGMP production in Purkinje cells in rat cerebellar cortex.

Nitric oxide activation of guanylyl cyclase in cells revisited

Nitric oxide (NO) elicits physiological effects in cells largely by activating guanylyl cyclase (GC)-coupled receptors, leading to cGMP accumulation. Like other receptor-coupled effector mechanisms, NO stimulation of GC activity was previously considered to be a graded, concentration-dependent response, with deactivation following swiftly once the agonist disappeared. Recently, a new and unconventional mechanism has been proposed from experiments on purified protein [Cary, S. P. L., Winger, J. A. & Marletta, M. A. (2005) Proc. Natl. Acad. Sci. USA 102, 13064-13069]. It was concluded that GC in vivo will display a dual regulation by NO: a long-lasting tonic activity (10-20% of maximum) due to persistent occupation by NO of the heme binding site and phasic activity due to engagement of another unidentified, lower affinity site. The hypothesis was first tested by monitoring GC activity in rat platelets maintained in vitro and exposed to calibrated NO transients. The kinetics was as expected for a single binding site for NO (EC(50) = 10 nM), with activation and deactivation of enzyme activity conforming to the predictions of a simple receptor model. No tonic GC activity attributable to long-term NO binding was detected after exposure to the full range of active NO concentrations (peaking at 2-500 nM). Comparable results were obtained by using neural cells isolated from the cerebellum. After exposure to high NO concentrations, persistent GC activity could be recorded, but this activity was caused artifactually by secondary NO sources being formed in the medium. The new scheme for regulation of GC activity by NO is of doubtful relevance to cells.

Chemopreventive alteration of the cell-cell adhesion in head and neck squamous cell cancer

Approximately 310,000 new cases of oral and pharynx cancer account for a major cause of neoplasm related morbidity and mortality world-wide. Unfortunately, the survival rate has not improved significantly in the last decade. The vast majority of head and neck cancer is squamous cell carcinoma. The major adhesion-proteins involved in the development and maintenance of all solid tissue are the Cadherins. Cadherins are the transmembrane components of the adherent junction with interaction with plakoglobin and beta-catenin. Downregulation of Cadherins and catenins is frequently observed in many types of human cancer. Sulindac sulfone is one of the new therapeutic apoptotic agents that show promise in the treatment of cancer. In this study, we incubated sulindac sulfone with a head and neck cancer cell line and investigated the outcome of E-Cadherin. Immunohistochemical and Western blot analyses were then performed, with different concentrations of sulindac sulfone (100, 200, 400, 600, and 800 microMol) for 48 h. At 400 microMol of sulindac sulfone a decrease of 21% was observed; at 600 microMol, 44% decrease of beta-catenin concentration was seen, and incubation with 800 microMol resulted in 73% reduction of secreted beta-catenin. Incubation with sulindac sulfone seemed to stop proliferation; however, with respect to the controls, there was no increased reduction of the total protein. Sulindac sulfone resulted in an increase of E-Cadherin content in the head and neck squamous cell cancer cell line after 48 h of incubation; however, the reactivity was restricted to the adherent junctions. At increasing concentrations of sulindac sulfone, intercellular E-Cadherin immunostaining intensifyied. ELISA also depicted significant rising levels of E-Cadherin. Sulindac sulfone contributes to the inactivation of cGMP phospho-diesterase. Thus, the accumulation of cellular cGMP and protein kinase G is induced. The following degradation of the phosphorylated beta-catenin and the dissociation from the Cadherin-catenin complex releases E-Cadherin. This may also contribute to growth inhibition and co-ordinate with apoptosis induction. It is not really clear as to, which pathway results in the elevation of the E-Cadherin proteins. However, in epithelial cancer cells, the Cadherin-catenin complex serves as a target for the chemopreventive agent, sulindac sulfone.

Mechanism of reactive oxygen species generation after opening of mitochondrial KATP channels

opening of the ATP-sensitive K+ (KATP) channel mediates the cardioprotective effect induced by pathophysiological stressors such as ischemic preconditioning (IPC) ([15][1], [29][2]), heat shock ([19][3]), and pharmacological agents, including adenosine ([5][4]), ACh ([26][5]), opioids ([12][6]),