Stimulated cGMP Production Research Articles

Oxidant-Redox Regulation of Pulmonary Vascular Responses to Hypoxia and Nitric Oxide-cGMP Signaling

Most current theories for the mechanism of hypoxic pulmonary vasoconstriction (HPV) include a role for reactive oxygen species and/or changes in redox regulation, but extreme controversy exists regarding which systems and redox changes mediate the HPV response. Nitric oxide (NO) appears to help to maintain low pulmonary arterial pressure, suppress HPV, and prevent the development of pulmonary hypertension. Our studies have found a key role for glucose-6-phosphate dehydrogenase in bovine pulmonary arterial smooth muscle functioning to maintain elevated levels of cytosolic NADPH which fuels the generation of vasodilator levels of hydrogen peroxide. HPV results from hypoxia removing vasodilation by peroxide. Decreased superoxide generation by Nox4 oxidase and its conversion to peroxide by Cu,Zn-SOD appear to be potential factors in sensing hypoxia, and decreased cGMP-associated vasodilation and removal of redox controlled vasodilator mechanisms by increased cytosolic NADPH may be key coordinators of the HPV response. Oxidant generation associated with vascular disease processes, including the removal of NO by superoxide, and attenuation of its ability to stimulate cGMP production by oxidation of the heme and thiols of soluble guanylate cyclase attenuate potential beneficial actions of NO on pulmonary arterial function. While pulmonary hypertension appears to have multiple poorly understood effects on redox-associated processes, potentially influencing responses to hypoxia and NO-cGMP signaling, much remains to be elucidated regarding how these processes may be important factors in the progression, expression and therapeutic treatment of pulmonary hypertension.

Cardiac Depression Produced by L-Arginine and Phosphodiestrase Inhibitor on Isolated Mammalian Rabbit Heart: Function of Cyclic Guanosine Monophosphate (cGMP)

Problem statement: Cyclic guanosine 3',5'-monophosphate cGMP is one the important second messengers that determines the cardiomyocyte activity and its role in healthy and diseased cardiac muscle is still controversial. We are reporting the effect of adding L-arginine, the NO donor that stimulates cGMP production and Sildenafil citrate (phosphodiestrase inhibitor) that inhibits cGMP hydrolyis on isolated rabbit's heart to answer: Is it safe to prescribe phosphodiestrase inhibitors for men with low cardiac output? Approach: Isolated hearts from 6 rabbits were perfused using Langendorff's apparatus in which the perfusion fluid was ringer-Locke solution, applied at constant flow rate and was continuously bubbled with a mixture of 95% oxygen and 5% carbon dioxide. Each heart served as its own control before infusion of L-arginine in concentration of 3 m mol L −1 and Sildenafil citrate 1.5 mg L −1 simultaneously. Their effects were recorded after 1, 3, 5 and 10 min. The effluent fluid was collected for cardiac enzymes assay after 5 and 10 min. Results: Data showed that the infusion of L-arginine and Sildenafil citrate produced negative inotrpic and chronotropic effects. Also, the cardiac enzymes were significantly elevated. Conclusion: The present study, which was carried out on the isolated rabbit's heart, demonstrated that increased cGMP could produce a cardioprotective role by decreasing the cardiac work, although it might be hazardous to men with depressed cardiac function.

Distinct Roles of Protease-Activated Receptors in Signal Transduction Regulation of Endothelial Nitric Oxide Synthase

Protease-activated receptors (PARs), such as PAR1 and PAR2, have been implicated in the regulation of endothelial NO production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase through the selective phosphorylation of a positive regulatory site, Ser(1179), and a negative regulatory site, Thr(497), in bovine aortic endothelial cells. A selective PAR1 ligand, TFLLR, stimulated the phosphorylation of endothelial NO synthase at Thr(497). It had a minimal effect on Ser(1179) phosphorylation. In contrast, a selective PAR2 ligand, SLIGRL, stimulated the phosphorylation of Ser(1179) with no noticeable effect on Thr(497). Thrombin has been shown to transactivate PAR2 through PAR1. Thus, thrombin, as well as a peptide mimicking the PAR1 tethered ligand, TRAP, stimulated phosphorylation of both sites. Also, thrombin and SLIGRL, but not TFLLR, stimulated cGMP production. A G(q) inhibitor blocked thrombin- and SLIGRL-induced Ser(1179) phosphorylation, whereas it enhanced thrombin-induced Thr(497) phosphorylation. In contrast, a G(12/13) inhibitor blocked thrombin- and TFLLR-induced Thr(497) phosphorylation, whereas it enhanced the Ser(1179) phosphorylation. Although a Rho-kinase inhibitor, Y27632, blocked the Thr(497) phosphorylation, other inhibitors that targeted Rho-kinase failed to block TFLLR-induced Thr(497) phosphorylation. These data suggest that PAR1 and PAR2 distinctly regulate endothelial NO synthase phosphorylation and activity through G(12/13) and G(q), respectively, delineating the novel signaling pathways by which the proteases act on protease-activated receptors to potentially modulate endothelial functions.

Abstract 1913: Distinct eNOS Regulation by Protease-Activated Receptor 1 and 2 Involving Gq, G12/13 and Y27632-Sensitive Non-Rho Kinase

Protease-activated receptors (PARs), such as PAR1 and PAR2 have been implicated in the regulation of endothelial nitric oxide (NO) production. We hypothesized that PAR1 and PAR2 distinctly regulate the activity of endothelial NO synthase (eNOS) through the selective phosphorylation of a positive regulatory site, Ser 1179 and a negative regulatory site, Thr 497 . In bovine aortic endothelial cells (BAECs), a PAR1 ligand, TFLLR, phosphorylated eNOS at Thr 497 . It had no effect on Ser 1179 phosphorylation or cyclic GMP (cGMP) production. In contrast, a PAR2 ligand, SLIGRL, phosphorylated Ser 1179 with no noticeable effect on Thr 497 . SLIGRL stimulated cGMP production that was blocked by L-NAME. Neither eNOS phosphorylation nor cGMP production was observed by a PAR4 agonist, AY-NH2. Thrombin has been shown to transactivates PAR2 through PAR1. Thus, thrombin stimulates eNOS phosphorylation at both sites as well as cGMP production in BAECs. Importantly, SLIGRL-induced Ser 1179 phosphory-lation and cGMP production were inhibited by a G q inhibitor, YM-254890. In addition, eNOS phosphorylation was not affected by over-expression of GRK2 C-tail or pertusis toxin pretreatment. By contrast, TFLLR-induced Thr 497 phosphorylation was selectively inhibited by a Rho-kinase inhibitor, Y27632, and over-expression of p115RhoGEF RGS domain, a selective G 12/13 inhibitor. However, other inhibitors (H-1152 and dnRho) that target Rho-kinase or a PKC inhibitor, GF109203X, failed to block TFLLR-induced Thr 497 phosphorylation. Taken together, these data suggest that PAR1-induced phosphorylation of eNOS at Thr 497 requires G 12/13 and subsequent activation of a Y27632-sensitive eNOS Thr 497 kinase that is distinct from Rho-kinase or PKC. We conclude that PAR1 and PAR2 distinctly regulate eNOS activity through G q and G 12/13 , respectively, delineating the novel signaling pathways by which proteases regulate eNOS activity. This research has received full or partial funding support from the American Heart Association, AHA National Center.

STa peptide analogs for probing guanylyl cyclase C

Guanylyl cyclase C (GC-C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18-residue heat-stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12-residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC-C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.

Two Autocrine Pathways to Regulate Cyclic GMP Synthesis in Cultured Human Retinal Pigment Epithelial Cells

Aim: To investigate the role of two separate enzymatic pathways [soluble (sGC) vs. particulate (pGC) guanylyl cyclase] in the synthesis of cyclic GMP (cGMP) in cultured human retinal pigment epithelial (RPE) cells. Methods: cGMP accumulation was evaluated by quantitative analysis of cGMP immunoreactivity. RPE cells were also stained for inducible nitric oxide synthase (iNOS), ANP and β₁- and α₂-subunits of sGC. Results: We showed nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and iNOS immunoreactivity in RPE cells. Incubation of the cells in the presence of 1 mM IBMX to inhibit phosphodiesterase activity, and the simultaneous inhibition of NOS activity with L-NAME suggested the involvement of sGC in maintaining a low level of cGMP in the RPE cells. The involvement of sGC was further supported by detection of the β₁- and α₂-subunits of sGC. Incubation of the cells in the presence of atrial natriuretic peptide (ANP) to stimulate pGC strongly increased cGMP immunoreactivity. We also demonstrated the presence of ANP in all RPE cells. Conclusion: Cultured human RPE cells are capable of producing cGMP after stimulation of sGC or pGC. The presence of iNOS and ANP in all cells suggests two different autocrine pathways of stimulating cGMP production in these cells. The possible role of cGMP in the regulation iNOS gene expression and in the regulation of ANP is discussed.

Nitric Oxide Inhibits Metamorphosis in Larvae ofCrepidula fornicata, the Slippershell Snail

This paper concerns the role of nitric oxide (NO) in controlling metamorphosis in the marine gastropod Crepidula fornicata. Metamorphosis was stimulated by the nitric oxide synthase (NOS) inhibitors AGH (aminoguanidine hemisulfate) and SMIS (S-methylisothiourea sulfate) at concentrations of about 100-1000 micromol l(-1) and 50-200 micromol l(-1), respectively. Metamorphosis was not, however, induced by the NOS inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) at even the highest concentration tested, 500 micromol l(-1). Moreover, pre-incubation with l-NAME at 20 and 80 micromol l(-1) did not increase the sensitivity of competent larvae to excess K(+), a potent inducer of metamorphosis in this species; we suggest that either l-NAME is ineffective in suppressing NO production in larvae of C. fornicata, or that it works only on the constitutive isoform of the enzyme. In contrast, metamorphosis was potentiated by the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) in response to a natural metamorphic inducer derived from conspecific adults. Because NO typically stimulates cGMP production through the activation of soluble guanylate cyclase, this result supports the hypothesis that NO acts as an endogenous inhibitor of metamorphosis in C. fornicata. The expression of NOS, shown by immunohistochemical techniques, was detected in the apical ganglion of young larvae but not in older larvae, further supporting the hypothesis that metamorphosis in C. fornicata is made possible by declines in the endogenous concentration of NO during development.

The receptor attributable to C‐type natriuretic peptide‐induced differentiation of osteoblasts is switched from type B‐ to type C‐natriuretic peptide receptor with aging

C-type natriuretic peptide (CNP) stimulates the differentiation and inhibits the proliferation of osteoblastic lineage cells. In this study, we examined whether the effects of CNP on osteoblastic functions change with aging using calvarial osteoblast-like cells from 25-week-old (young) and 120-week-old (aged) rats. CNP inhibited DNA synthesis and stimulated collagen synthesis and mineralized bone nodule formation. These effects were less pronounced in aged rat cells, suggesting the age-related attenuation of CNP-induced signaling. They were also blocked by the treatment of young rat cells with KT5823, a protein kinase G (PKG) inhibitor, but not by the treatment of aged rat cells with KT5823. CNP stimulated cGMP production in young rat cells, but not in aged rat cells. Natriuretic peptide receptor (NPR)-B, which has a guanylyl cyclase activity domain, and NPR-C, which has no enzyme activity domain, were predominantly expressed in young and aged rat cells, respectively. C-ANF, an NPR-C agonist, mimicked the effects of CNP on the proliferation and differentiation of aged rat cells; these effects were inhibited by the treatment with pertussis toxin (PTX), a Gi protein inhibitor. CNP and C-ANF evoked intracellular levels of inositol-1,4,5-triphosphate and Ca(2+), which are markers for phospholiase C (PLC) activation, in aged rat cells, and the effects of these two peptides were also blocked by the treatment with PTX. From these results, we concluded that CNP acts as a positive regulator of bone formation by osteoblasts and that the signaling pathway for CNP is switched from NPR-B/cGMP/PKG to NPR-C/G(i) protein/PLC with aging.

Insulin-inhibited and stimulated cultured vascular smooth muscle cell migration are related to divergent effects on protein phosphatase-2A and autonomous calcium/calmodulin-dependent protein kinase II

Insulin, in the permissive presence of nitric oxide (NO), stimulates cGMP production which inhibits autonomous calcium/calmodulin-dependent protein kinase II (CaM kinase II) thereby inhibiting cultured vascular smooth muscle cell (VSMC) migration. In the presence of angiotensin II (Ang II), insulin stimulates NAD(P)H oxidase activity leading to increased VSMC migration. We wished to see whether insulin-stimulated cGMP stimulates protein phosphatase-2A (PP-2A) thereby inhibiting autonomous CaM kinase II and migration, and whether insulin, in the presence of Ang II, inhibits PP-2A and stimulates autonomous CaM kinase II in a NAD(P)H oxidase-dependent manner. One nanomole per litre of insulin in the presence of NO, or 50μmol/L 8-Br-cGMP stimulated PP-2A activity by 46±6 and 247±23%, respectively (both P<0.05), and 8-Br-cGMP inhibited autonomous CaM kinase II activity by 67±9% (P<0.05) by a 10nmol/L okadaic acid-sensitive pathway. Insulin plus Ang II inhibited PP-2A activity by 57±7% (P<0.05) and stimulated autonomous CaM kinase II activity by 120±14% (P<0.05), both by an apocynin-sensitive pathway. 8-Br-cGMP-inhibited VSMC migration was blocked by okadaic acid. It is concluded that insulin in the presence of NO stimulates cGMP which stimulates PP-2A activity causing inhibition of autonomous CaM kinase II activity and thus VSMC migration, and that insulin in the presence of Ang II inhibits PP-2A and stimulates autonomous CaM kinase II activities by a NAD(P)H oxidase-dependent mechanism which are associated with insulin-stimulated NAD(P)H oxidase-dependent migration.

Dendroaspis natriuretic peptide binds to the natriuretic peptide clearance receptor

Dendroaspis natriuretic peptide (DNP) is a newly-described natriuretic peptide which lowers blood pressure via vasodilation. The natriuretic peptide clearance receptor (NPR-C) removes natriuretic peptides from the circulation, but whether DNP interacts with human NPR-C directly is unknown. The purpose of this study was to test the hypothesis that DNP binds to NPR-C. ANP, BNP, CNP, and the NPR-C ligands AP-811 and cANP(4–23) displaced [ 125I]-ANP from NPR-C with pM-to-nM K i values. DNP displaced [ 125I]-ANP from NPR-C with nM potency, which represents the first direct demonstration of binding of DNP to human NPR-C. DNP showed high pM affinity for the GC-A receptor and no affinity for GC-B ( K i > 1000 nM). DNP was nearly 10-fold more potent than ANP at stimulating cGMP production in GC-A expressing cells. Blockade of NPR-C might represent a novel therapeutic approach in augmenting the known beneficial actions of DNP in cardiovascular diseases such as hypertension and heart failure.

Nitric Oxide-Independent cGMP Efflux in the Striatum of Rats Exposed to Carbon Monoxide as Determined by Microdialysis

Extracellular cGMP in the striatum of rats exposed to 3000 ppm carbon monoxide (CO) or 8% O2 was decreased during the early period of exposure. Thereafter, extracellular cGMP in rats exposed to CO, but not 8% O2, was transiently increased. A nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine, strongly reduced the steady-state level of extracellular cGMP in the striatum, indicating a primary role of NO in cGMP production. However, it failed to suppress the CO-induced increase in extracellular cGMP in the striatum. These findings suggest that CO may stimulate cGMP production in rat striatum independently of NO and hypoxia.

Activation of Endothelial Nitric Oxide Synthase by the Angiotensin II Type 1 Receptor

Enhanced angiotensin II (AngII) action has been implicated in endothelial dysfunction that is characterized as decreased nitric oxide availability. Although endothelial cells have been reported to express AngII type 1 (AT1) receptors, the exact role of AT1 in regulating endothelial NO synthase (eNOS) activity remains unclear. We investigated the possible regulation of eNOS through AT1 in bovine aortic endothelial cells (BAECs) and its functional significance in rat aortic vascular smooth muscle cells (VSMCs). In BAECs infected with adenovirus encoding AT1 and in VSMCs infected with adenovirus encoding eNOS, AngII rapidly stimulated phosphorylation of eNOS at Ser1179. This was accompanied with increased cGMP production. These effects were blocked by an AT1 antagonist. The cGMP production was abolished by a NOS inhibitor as well. To explore the importance of eNOS phosphorylation, VSMCs were also infected with adenovirus encoding S1179A-eNOS. AngII did not stimulate cGMP production in VSMCs expressing S1179A. However, S1179A was able to enhance basal NO production as confirmed with cGMP production and enhanced vasodilator-stimulated phosphoprotein phosphorylation. Interestingly, S1179A prevented the hypertrophic response similar to wild type in VSMCs. From these data, we conclude that the AngII/AT1 system positively couples to eNOS via Ser1179 phosphorylation in ECs and VSMCs if eNOS and AT1 coexist. However, basal level NO production may be sufficient for prevention of AngII-induced hypertrophy by eNOS expression. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for eNOS gene therapy under endothelial dysfunction.

Lysophosphatidylcholine stimulation of CGMP production in human endothelial cells involves tyrosine kinases, heterotrimeric GTP binding proteins, phosphatidylinositol 3-kinase and protein kinase C

Lysophosphatidylcholine stimulation of CGMP production in human endothelial cells involves tyrosine kinases, heterotrimeric GTP binding proteins, phosphatidylinositol 3-kinase and protein kinase C

Identification of two functional guanylin receptors in eel: Multiple hormone-receptor system for osmoregulation in fish intestine and kidney

Guanylyl cyclase C (GC-C) is a single transmembrane receptor for a family of intestinal hormones, guanylins. In the eel, we previously identified three guanylins, whose gene expression was enhanced in the intestine after transfer from fresh water to seawater. However, only limited information is available about the structure and function of their receptor(s). In the present study, we cloned full-length cDNAs encoding two isoforms of GC-C, named GC-C1 and GC-C2, from eel intestine. The predicted GC-C proteins consisted of extracellular ligand-binding domain, membrane-spanning domain, kinase-like domain and cyclase catalytic domain, in which GC-C-specific sequences were largely conserved. Phylogenetic analyses showed that the cloned membrane GCs are grouped with the GC-C of other vertebrates but not with GC-A and GC-B. However, eel GC-Cs appear to have undergone unique structural evolution compared with other GC-Cs. The three eel guanylins (guanylin, uroguanylin and renoguanylin), but not eel atrial natriuretic peptide, stimulated cGMP production dose-dependently in COS cells expressing either of the cloned cDNAs, providing functional support for assignment as eel guanylin receptors. The potency order for cGMP production was uroguanylin > guanylin ⩾ renoguanylin for GC-C1; guanylin ⩾ renoguanylin > uroguanylin for GC-C2. The distinctive ligand selectivity was consistent with the low homology (53%) of the extracellular domain of the two GC-Cs compared with that observed for other domains (74–90%). Both GC-C genes were expressed in the alimentary tract (esophagus, stomach and intestine) and kidney, and their expression was higher in the intestine of seawater-adapted eels than that of freshwater eels just as observed with the guanylin genes. However, the expression of the receptor genes was unchanged for 24 h after transfer of eels from fresh water to seawater or vice versa, showing slower response of the receptors to salinity changes than their ligands. Collectively, the multiple guanylin—GC-C system may be involved as a paracrine factor in seawater adaptation at the intestine and kidney of the eel.

Chickens Express Only Uroguanylin for Regulation of cGMP Production

Genes encoding uroguanylin and guanylin are found in all vertebrates that have been investigated thus far. Both peptides activate a receptor-guanylate cyclase-C (R-GC-C) signaling molecule. In this study, cDNAs were isolated encoding chicken forms of preprouroguanylin and R-GC-C. A bioactive uroguanylin peptide is located at the C-terminus of a 110 residue precursor. Chicken uroguanylin stimulates cGMP production in human T84 intestinal and opossum kidney cells. Uroguanylin genes are located on chromosome 21 and genes encoding R-GC-C on chromosome 1 of Gallus gallus. Uroguanylin alone regulates the enzymatic activity of R-GC-C in chickens because no evidence for genes encoding preproguanylin was found in the database from the Chicken Genone project. Transcripts for uroguanylin were detected in the small and large intestine, liver, pancreas, fat, uterus, magnum and heart. R-GC-C mRNAs were detected in the intestine, pancreas, fat, magnum and kidney. Administration of oral NaCl increases mRNA levels for uroguanylin and R-GC-C in the intestine, kidney and magnum segment of oviduct. Therefore, chickens express uroguanylin alone and no guanylin for regulation of R-GC-C enzyme activity and cGMP production in target organs. Dietary salt influences both uroguanylin and R-GC-C mRNAs in tissues involved in sodium balance like the intestine and kidney as well as novel tissues such as the magnum segment of avian oviduct.

Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation

AbstractLocal vasodilation in response to hypoxia is a fundamental physiologic response ensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release. NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (P50). Concomitantly, vasodilation is initiated at the P50, suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation of vasodilation. Mutation of the conserved β93cys residue decreases the heme redox potential (ie, decreases E1/2), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose “enzyme activity” couples hypoxia to increased NO-dependent blood flow.

Peripheral N-Methyl-d-Aspartate Receptors Modulate Nonadrenergic Noncholinergic Lower Esophageal Sphincter Relaxation in Rabbits

We investigated the role of peripheral N-methyl-D-aspartate (NMDA) receptors in the myenteric plexus in mediating nonadrenergic noncholinergic (NANC) nitrergic relaxation of the lower esophageal sphincter (LES). Isometric contraction of LES strips from Japanese White rabbits was measured. NANC relaxation was induced by KCl (30 mM) in the presence of atropine and guanethidine. The concentration of 3',5'-cyclic guanosine monophosphate (cGMP) was measured using a radioimmunoassay. The muscle strips were exposed to diethyldithiocarbamic acid (DETCA; 3 mM) to inactivate Cu/Zn superoxide dismutase. MK801 (5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine) inhibited NANC relaxation in a concentration-dependent manner (EC50 = 1.5 x 10(-5) M), accompanied by a decrease in cGMP production. NMDA induced a concentration-dependent relaxation, which was antagonized by MK801. NMDA stimulated cGMP production, which was inhibited by N(G)-nitro-L-arginine. Superoxide dismutase (100 U/mL) shifted the concentration-response relationship of MK801-mediated inhibition of NANC relaxation to the right (EC50 = 3.4 x 10(-5) M), whereas catalase did not. Treatment with DETCA shifted the concentration-response relationships of pyrogallol-, ketamine- and MK801-mediated inhibition of NANC relaxation to the left. These findings suggest that the peripheral NMDA receptors mediate NANC smooth muscle relaxation, and modulate it, in part, through extracellular production of superoxide anions, thus eliminating the relaxant effect of endogenous nitric oxide.

Dual actions of dephostatin on the nitric oxide/cGMP-signalling pathway in porcine iliac arteries

We examined the effects of the nitrosoamine dephostatin on the nitric oxide (NO)/cyclic guanosine 3′,5′-monophosphate (cGMP)-signalling in porcine iliac arteries. Dephostatin has been characterised as a tyrosine phosphatase inhibitor, but Western blot analyses showed that dephostatin did not augment tyrosine phosphorylation of arterial proteins. However, dephostatin relaxed pre-contracted arteries, and this effect was antagonised by the soluble guanylyl cyclase inhibitor 1 H[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ). Furthermore, dephostatin increased the cGMP content and the serine phosphorylation of vasodilator-stimulated phosphoprotein. Dephostatin also inhibited the relaxation induced by acetylcholine and the NO-donor S-nitroso- N-acetyl-penicillamine (SNAP). In contrast, dephostatin did not affect the NO-dependent actions of 1,2,3,4-Oxatriazolium, 3-(3-chloro-2-metylphenyl)-5-[[(4methylphenyl)sulfonyl]amino]-hydroxide inner salt (GEA 3175). Measurement of NO revealed that dephostatin accelerated the consumption of NO. In conclusion, dephostatin exerts dual effects on the NO/cGMP-signalling pathway in iliac arteries. The drug actions included scavenging of NO, but also stimulation of cGMP production. These effects were not related to inhibition of tyrosine phosphatases.

Signal transduction pathways involved in particulate matter induced relaxation in rat aorta—Spontaneous hypertensive versus Wistar Kyoto rats

Previously we reported that in vivo exposure to ambient particulate matter (PM) induces vasodilatation in rat aorta. The purpose of the current study was to investigate the intracellular messengers involved in PM-elicited vasodilatation in aortas from spontaneous hypertensive (SHR) and normotensive (WKY) rats. MethodsThe contribution of three different intracellular pathways, i.e. (1) the NO-cGMP pathway, (2) prostanoids signaling and (3) endothelial hyperpolarisation factors were evaluated by using specific inhibitors (NS2028, Diclofenac and high K-concentration/17-ODYA, respectively). Using antagonists of capsaicin- or histamine receptors we tested potential interactions of PM with these receptors. Particle suspensions (EHC-93), particle filtrates (particle-free) and Cu2+- or Zn2+-containing solutions were used to obtain cumulative dose–response curves of relaxation in normal and endothelium-denuded rings. ResultsOur present data confirm that PM and its soluble components elicit an endothelium-independent vasodilatation in rat aorta rings. The response is mainly linked to the activation of soluble guanylate cyclase (sGC), since its inhibition by NS2028 almost abolished relaxation. Indeed PM suspensions stimulated cGMP production in purified isolated sGC. Neither the receptor nor their signaling pathways played a significant role in the direct relaxation by PM or metals. Vasodilatation responses were significantly higher in SHR than WKY control rats. ConclusionOur data demonstrate that PM elicits a dose-dependent vasodilatation via activation of sGC in vascular smooth muscles. PM components, including soluble transition metals play a major role in this response. The stronger effect in SHR rats is in accordance with the observation that acute effects of PM are mainly seen in patients with underlying cardiovascular diseases.

Cardiac-specific attenuation of natriuretic peptide A receptor activity accentuates adverse cardiac remodeling and mortality in response to pressure overload

Atrial (ANP) and brain (BNP) natriuretic peptides are hormones of myocardial cell origin. These hormones bind to the natriuretic peptide A receptor (NPRA) throughout the body, stimulating cGMP production and playing a key role in blood pressure control. Because NPRA receptors are present on cardiomyocytes, we hypothesized that natriuretic peptides may have direct autocrine or paracrine effects on cardiomyocytes or adjacent cardiac cells. Because both natriuretic peptides and NPRA gene expression are upregulated in states of pressure overload, we speculated that the effects of the natriuretic peptides on cardiac structure and function would be most apparent after pressure overload. To attenuate cardiomyocyte NPRA activity, transgenic mice with cardiac specific expression of a dominant-negative (DN-NPRA) mutation (HCAT D 893A) in the NPRA receptor were created. Cardiac structure and function were assessed (avertin anesthesia) in the absence and presence of pressure overload produced by suprarenal aortic banding. In the absence of pressure overload, basal and BNP-stimulated guanylyl cyclase activity assessed in cardiac membrane fractions was reduced. However, systolic blood pressure, myocardial cGMP, log plasma ANP levels, and ventricular structure and function were similar in wild-type (WT-NPRA) and DN-NPRA mice. In the presence of pressure overload, myocardial cGMP levels were reduced, and ventricular hypertrophy, fibrosis, filling pressures, and mortality were increased in DN-NPRA compared with WT-NPRA mice. In addition to their hormonal effects, endogenous natriuretic peptides exert physiologically relevant autocrine and paracrine effects via cardiomyocyte NPRA receptors to modulate cardiac hypertrophy and fibrosis in response to pressure overload.