Impaired Redox and Protein Homeostasis as Risk Factors and Therapeutic Targets in Toxin-Induced Biliary Atresia.

BACKGROUND & AIMS: Extrahepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA. METHODS: We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screening of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model, with subsequent validation. RESULTS: Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors and other cyclic guanosine monophosphate signaling activators worked synergistically with the glutathione precursor N-acetylcysteine in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. Phosphodiesterase-5 inhibitors enhanced proteasomal degradation and required intact HSP90 chaperone. CONCLUSION: Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone and may account for the reported association between BA transplant-free survival and glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with N-acetylcysteine and cyclic guanosine monophosphate signaling enhancers warrants further investigation as therapy for BA.

Molecular Mechanisms of Phosphodiesterase-5 Inhibitors on Neuronal Apoptosis.

Phosphodiesterase-5 inhibitors (PDE5Is) have been shown to modulate cell death/cell survival in different in vivo and in vitro models of disease by activating many signaling pathways. This review aimed at elucidating how PDE5Is can inhibit apoptosis. In this study, we describe many signaling pathways involved with the mechanism of action of PDE5Is that ultimately inhibit apoptosis and thus promote cell survival.

Pharmacokinetic and Pharmacodynamic Profiles of Glyco-Modified Atrial Natriuretic Peptide Derivatives Synthesized Using Chemo-enzymatic Synthesis Approaches.

Atrial natriuretic peptide (ANP) exerts beneficial pharmacological effects in the treatment of various cardiovascular disorders, such as acute congestive heart failure (ADHF). However, the clinical use of ANP is limited to the continuous intravenous infusion owing to its short half-life (2.4 ± 0.7 min). In the present study, we conjugated the glyco-modified ANP with a monoclonal antibody (mAb) or an Fc via chemo-enzymatic glyco-engineering using EndoS D233Q/Q303L. The most potent derivative SG-ANP-Fc conjugate extended the half-life to 14.9 d and the duration of blood pressure lowering effect to over 28 d. This new biologic modality provides an opportunity to develop outpatient therapy after ADHF.

Exenatide induces an increase in vasodilatory and a decrease in vasoconstrictive mediators.

In view of the known vasodilatory effects of glucagon-like peptide-1 and exenatide, we investigated the effects of exenatide on vasoactive factors. We analysed blood samples and mononuclear cells (MNCs) from a previous study, collected after a single dose and 12 weeks of exenatide or placebo treatment in a series of 24 patients with type 2 diabetes mellitus. After exenatide treatment, plasma concentrations of atrial natriuretic peptide, cyclic guanyl monophosphate (cGMP) and cyclic adenyl monophosphate increased significantly at 12 weeks. Plasma cGMP and adenylate cyclase expression in MNCs increased significantly after a single dose. Angiotensinogen concentration fell significantly 2 hours after a single dose and at 12 weeks, while renin and angiotensin II levels fell significantly only after a single dose and not after 12 weeks of treatment. Exenatide also suppressed the plasma concentration of transforming growth factor-ß and the expression of P311 in MNCs at 12 weeks. Thus, exenatide induces an increase in a series of vasodilators, while suppressing the renin-angiotensin system. These changes may contribute to the overall vasodilatory effect of exenatide.

Carbon monoxide reverses adipose tissue inflammation and insulin resistance upon loss of ovarian function.

We hypothesized that carbon monoxide (CO) might suppress chronic inflammation, which led to metabolic disturbances. Ovariectomy (OVX) was performed in mice to mimic chronic inflammation secondary to loss of ovarian function. OVX increased fat mass and the infiltration of highly inflammatory CD11c cells into adipose tissue (AT), resulting in a disturbance of glucose metabolism. Treatment of CO attenuated these; CO decreased recruitment of CD11c-expressing cells in AT and reduced expression of CD11c in bone marrow-derived macrophages, protecting them from M1 polarization. Upregulated cGMP and decreased reactive oxygen species were responsible for the inhibitory activity of CO on CD11c expression; knockdown of soluble guanylate cyclase or heme oxygenase-1 using small interfering RNAs reduced this inhibition substantially. Improved OVX-induced insulin resistance (IR) by CO was highly associated with its activity to attenuate AT inflammation. Our results suggest a therapeutic value of CO to treat postmenopausal IR by reducing AT inflammation.

Role of both actin-myosin cross bridges and NO-cGMP pathway modulators in the contraction and relaxation of human placental stem villi.

INTRODUCTION: Human placental stem villi (PSV) present contractile properties. We studied the role of actin-myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation. METHODS: In vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL. RESULTS: PSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups. DISCUSSION AND CONCLUSION: Isolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes.

Cyclic GMP induced apoptosis via protein kinase G in oestrogen receptor-positive and -negative breast cancer cell lines.

The activation of protein kinase G (PKG) by cyclic guanosine 3,5-monophosphate (cGMP) has become of considerable interest as a novel molecular approach for the induction of apoptosis in cancer cells. The present study was designed to examine the effects of cGMP and PKG on cell growth and apoptosis in the human breast cancer cell lines, MCF-7 and MDA-MB-468. To achieve this, 1-benzyl-3-(5P-hydroxymethyl-2P-furyl) indazole (YC-1), a soluble guanylyl cyclase activator, and 8-bromo-cGMP (8-br-cGMP), a membrane-permeant and phosphodiesterase-resistant analogue of cGMP, were employed in MCF-7 and MDA-MB-468 cells. Then, the role of PKG in the induction of apoptosis was evaluated using KT5823 and Rp-8-pCPT-cGMP as specific inhibitors of PKG. The expression of PKG isoforms in these cell lines was also investigated. KT5823 and Rp-8-pCPT-cGMP significantly attenuated the loss of cell viability caused by YC-1 and 8-br-cGMP in these cells. This study provides direct evidence that the activation of PKG by cGMP induces growth inhibition and apoptosis in MCF-7 and MDA-MB-468 breast cancer cell lines.

Phosphodiesterases and cardiac cGMP: evolving roles and controversies.

cGMP and its primary target kinase, protein kinase G (PKG), are well recognized modulators of cardiac function and the chronic stress response. Their enhancement appears to serve as a myocardial brake, reducing maladaptive hypertrophy, improving cell survival, signaling and mitochondrial function, protecting against ischemia/reperfusion injury, and blunting the stimulatory effects of catecholamines. Translation of these effects into a chronic treatment for patients with heart failure based on increasing the generation of cGMP has been difficult, however, with tolerance and hypotension effects occurring with nitrates and neutral responses to natriuretic peptides (at least B-type). Inhibition of cGMP-targeted phosphodiesterases (PDEs) such as PDE5A is an alternative approach that appears to have more potent effects. Recent studies in experimental models and patients are revealing benefits in heart failure syndromes, and ongoing multicenter trials are testing the efficacy of PDE5A inhibition. In this review we discuss recent research findings and controversies regarding the PDE/cGMP/PKG signaling pathway, and suggest directions for further research.

A new approach to the treatment of cerebral vasospasm: the angiographic effects of tadalafil on experimental vasospasm.

BACKGROUND: The pathogenesis of cerebral vasospasm is likely to be multifactorial. Strong evidence has indicated that decreasing levels of NO after SAH seem to be important. A PDE-V inhibitor, tadalafil, theoretically increases NO levels. Our study investigated the vasodilatory efficacy of tadalafil on the cerebral arteries with measurement of basilar artery diameters on angiography. METHODS: We used 42 male Wistar-Albino rats to test our hypothesis. They were assigned randomly into the following seven groups: group 1: control (only saline), group 2: SAH only (killed on day 2), group 3: SAH + tadalafil (killed on day 2), group 4: SAH only (killed on day 4), group 5: SAH + tadalafil (killed on day 4), group 6: saline + tadalafil (killed on day 2) and group 7: saline + tadalafil (killed on day 4). The three different parts of basilar artery diameters were measured angiographically. RESULTS: There were statistically significant differences between the SAH and SAH groups treated with tadalafil at days 2 and 4. Comparison between control and tadalafil groups showed no significant differences. This result indicated that tadalafil has a vasodilatory effect on vasoconstricted arteries, but no effect on normal basilar arteries. CONCLUSION: Our study results showed that tadalafil has a vasodilatory effect on both acute and chronic periods of cerebral vasospasm. We also concluded that cerebral angiography can be used safely for investigation of cerebral vasospasm in animal studies.

D-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance.

In this paper, the role of D-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous D-Asp were detected, along with aspartate racemases that convert D-Asp to L-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered D-Asp. D-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since D-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that D-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

Nitric oxide upregulates dimethylarginine dimethylaminohydrolase-2 via cyclic GMP induction in endothelial cells.

Dimethylarginine dimethylaminohydrolase (DDAH) is an enzyme that metabolizes asymmetrical N(G),N(G)-dimethyl-L-arginine (ADMA) and N(G)-monomethyl-L-arginine (MMA), which are competitive endogenous inhibitors of NO synthase. However, it remains unknown whether NO itself influences DDAH activity and/or ADMA/MMA contents to regulate NO generation via a biofeedback mechanism. The present study was designed to examine the effects of NO on intracellular ADMA and MMA contents and DDAH gene expression levels and enzymatic activities in cultured rat aortic endothelial cells. The NO donors SNAP and NOR3 did not influence DDAH-1 expression but increased DDAH-2 mRNA and protein levels in concentration-dependent manners. SNAP upregulated DDAH enzymatic activity and reduced the MMA and ADMA contents but did not affect the symmetrical N(G),N'(G)-dimethyl-L-arginine and L-arginine levels, thereby negating a mediatory role for system y(+) in ADMA/MMA downregulation. The cGMP agonists 8-bromo-cGMP and C-type natriuretic peptide also stimulated DDAH-2 gene and protein expression levels and DDAH activity and increased the amount of nitrite/nitrate released into the culture supernatants. SNAP-induced DDAH-2 gene expression and DDAH activity were significantly inhibited by a protein kinase G inhibitor, KT5823, and a soluble guanylate cyclase inhibitor, ODQ, suggesting a mediatory role for cGMP in NO-induced DDAH-2 expression. Suppression of DDAH-2 mRNA using small interfering RNA technology abrogated NO-induced DDAH-2 expression. These data demonstrate that NO acts on endothelial cells to induce DDAH-2 expression via a cGMP-mediated process to reduce ADMA/MMA. Thus, the DDAH-2-ADMA/MMA-endothelial NO synthase regulatory pathway and NO-induced cGMP constitute a positive feedback loop that ultimately serves to maintain NO levels in the endothelial environment.

Nitric oxide stimulation of cGMP accumulation in myometrial cells from pregnant women is antagonized by oxytocin.

The role of cGMP in the myometrium of pregnant women is not completely known. We have previously shown in guinea pig, monkey and man that NO-induced relaxation of oxytocin-induced contractions is independent of cGMP accumulation. To approach an understanding of the role of cGMP in myometrium, we have developed smooth muscle cell cultures from pregnant women undergoing caesarian section at term. Cells, grown in standard media containing progesterone, express smooth muscle cell markers and are used within five doublings. Cells stimulated with NO donors increase their cGMP levels nearly 100 fold (basal = approximately 9 pmol/mg protein). In the presence of oxytocin (OT; 1 microM), cGMP accumulation in the presence of NO (100 microM) is significantly blunted (25 fold). Cyclic GMP-degradation is inhibited by the presence of the phosphodiesterase inhibitor zaprinast; suggesting that the ability of OT to attenuate cGMP accumulation is unlikely to be due to degradation. We propose that the elevation of intracellular calcium following the addition of OT suppress the activity of a calcium-sensitive guanylyl cyclase. The diminution of cGMP synthetic potential in myometrial cells from pregnant women is consistent with the absence of a role for cGMP in the NO-induced relaxation of uterine muscle.

Dissociation of dorsal root ganglion neurons induces hyperexcitability that is maintained by increased responsiveness to cAMP and cGMP.

Injury or inflammation affecting sensory neurons in dorsal root ganglia (DRG) causes hyperexcitability of DRG neurons that can lead to spontaneous firing and neuropathic pain. Recent results indicate that after chronic compression of DRG (CCD treatment), both hyperexcitability of neurons in intact DRG and behaviorally expressed hyperalgesia are maintained by concurrent activity in cAMP-protein kinase A (PKA) and cGMP-protein kinase G (PKG) signaling pathways. We report here that when tested under identical conditions, dissociation produces a pattern of hyperexcitability in small DRG neurons similar to that produced by CCD treatment, manifest as decreased action potential (AP) current threshold, increased AP duration, increased repetitive firing to depolarizing pulses, increased spontaneous firing and resting depolarization. A novel feature of this hyperexcitability is its early expression-as soon as testing can be conducted after dissociation (approximately 2 h). Both forms of injury increase the electrophysiological responsiveness of the neurons to activation of cAMP-PKA and cGMP-PKG pathways as indicated by enhancement of hyperexcitability by agonists of these pathways in dissociated or CCD-treated neurons but not in control neurons. Although inflammatory signals are known to activate cAMP-PKA pathways, dissociation-induced hyperexcitability is unlikely to be triggered by signals released from inflammatory cells recruited to the DRG because of insufficient time for recruitment during the dissociation procedure. Inhibition by specific antagonists indicates that continuing activation of cAMP-PKA and cGMP-PKG pathways is required to maintain hyperexcitability after dissociation. The reduction of hyperexcitability by blockers of adenylyl cyclase and soluble guanylyl cyclase after dissociation suggests a continuing release of autocrine and/or paracrine factors from dissociated neurons and/or satellite cells, which activate both cyclases and help to maintain acute, injury-induced hyperexcitability of DRG neurons.

The nitric oxide-cGMP signaling pathway differentially regulates presynaptic structural plasticity in cone and rod cells.

Although abundant structural plasticity in the form of axonal retraction, neurite extension, and formation of presynaptic varicosities is displayed by photoreceptors after retinal detachment and during genetic and age-related retinal degeneration, the mechanisms involved are mostly unknown. We demonstrated recently that Ca(2+) influx through cGMP-gated channels in cones and voltage-gated L-type channels in rods is required for neurite extension in vitro (Zhang and Townes-Anderson, 2002). Here, we report that the nitric oxide (NO)-cGMP signaling pathway is active in photoreceptors and that its manipulation differentially regulates the structural plasticity of cone and rod cells. The NO receptor soluble guanylyl cyclase (sGC) was detected immunocytochemically in both cone and rod cells. Stimulation of sGC increased cGMP production in retinal cultures. In cone cells, quantitative analysis showed that NO or cGMP stimulated neuritic sprouting; this stimulatory effect was dependent on both Ca2+ influx through cGMP-gated channels and phosphorylation by protein kinase G (PKG). At the highest levels of cGMP, however, cone outgrowth was no longer increased. In rod photoreceptors, NO or cGMP consistently inhibited neuritic growth in a dose-dependent manner; this inhibitory effect required PKG. When NO-cGMP signaling was inhibited, changes in the neuritic development of cone and rod cells were also observed but in the opposite direction. These results expand the role of cGMP in axonal activity to adult neuritogenesis and suggest an explanation for the neurite sprouting observed in an autosomal recessive form of retinitis pigmentosa that is characterized by high cGMP levels in photoreceptor layers.

Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor.

BACKGROUND & AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) provides an important apical route for Cl(-) secretion across intestinal epithelia. A thiazolidinone-type CFTR blocker (CFTR(inh)-172) reduced cholera toxin-induced fluid accumulation in mouse intestinal loops. Here, we characterize the efficacy and pharmacodynamics of CFTR(inh)-172 in blocking cAMP and cGMP induced Cl(-)/fluid secretion in rodent and human intestine. METHODS & RESULTS: CFTR(inh)-172 inhibited cAMP and cGMP agonist induced short-circuit current by >95% in T84 colonic epithelial cells (K(I) approximately 3 micromol/L) and in mouse and human intestinal sheets (K(I) approximately 9 micromol/L). A single intraperitoneal injection of CFTR(inh)-172 (200 microg) blocked intestinal fluid secretion in a rat closed-loop model by >90% for cholera toxin and >70% for STa Escherichia coli toxin. In mice, CFTR(inh)-172 (20 microg) inhibited cholera toxin-induced intestinal fluid secretion by 90% (persistence t(1/2) approximately 10 hours, K(I) approximately 5 microg) and STa toxin by 75% (K(I) approximately 10 microg). Tissue distribution and pharmacokinetic studies indicated intestinal CFTR(inh)-172 accumulation facilitated by enterohepatic circulation. An oral CFTR(inh)-172 preparation reduced fluid secretion by >90% in a mouse open-loop cholera model. CONCLUSIONS: A small molecule CFTR blocker markedly reduced intestinal ion and fluid secretion caused by cAMP/cGMP-dependent bacterial enterotoxins. CFTR inhibition may thus reduce fluid secretion in infectious secretory diarrheas.

Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum.

The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occasionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 microM) and by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1-100 microM) produced a concentration-dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and L-NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3',5'-cyclic monophosphate (0.1-100 microM) reduced in a concentration-dependent manner the spontaneous mechanical activity and at 100 microM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse duodenum and that guanylate cyclase stimulation and opening of Ca(2+)-dependent K(+) channels are independent mechanisms working in parallel to mediate NO action.

Activation and potentiation of the NO/cGMP pathway by NG-hydroxyl-L-arginine in rabbit corpus cavernosum under normoxic and hypoxic conditions and ageing.

1 When nitric oxide synthase (NOS) produces NO from N(G)-hydroxy-L-arginine (OH-arginine) instead of L-arginine, the total requirement of molecular oxygen and NADPH to form NO is reduced. The aim of this work was to evaluate the effects of OH-arginine on the contractility of rabbit corpus cavernosum (RCC) and to compare the capacities of L-arginine and OH-arginine to enhance NO-mediated responses under normoxic and hypoxic conditions and in ageing, as models of defective NO production. 2 OH-arginine, but not L-arginine, was able to relax phenylephrine-contracted rabbit trabecular smooth muscle. OH-arginine-induced relaxation was inhibited by the NOS-inhibitor, L-NNA (300 microM), and by the guanylyl cyclase inhibitor, ODQ (20 microM), while it was not affected by the cytochrome P450 oxygenase inhibitor, miconazole (0.1 mM). Administration of OH-arginine, but not L-arginine, produced a significant increment of cGMP accumulation in RCC tissue. 3 Relaxation elicited by OH-arginine (300 microM) was still observed at low oxygen tension. The increase of cGMP levels induced by ACh (30 microM) in RCC was significantly enhanced by addition of OH-arginine (300 microM) in normoxic conditions, as well as under hypoxia, while L-arginine did not alter the effects of ACh on cGMP accumulation. 4 Endothelium-dependent and nitrergic nerve-mediated relaxations were both significantly reduced in RCC from aged animals (>20-months-old) when compared with young adult rabbits (5-months-old). Treatment with OH-arginine (300 microM) significantly potentiated endothelium-dependent and neurogenic relaxation in corpus cavernosum from aged rabbits, while L-arginine (300 microM) did not have significant effects. 5 Results show that OH-arginine promotes NO-mediated relaxation of RCC and potentiates the NO-mediated responses induced by stimulation of endogenous NO generation in hypoxic and aged tissues. We propose that the use of OH-arginine could be of interest in the treatment of erectile dysfunction, at least in those secondary to defective NO production.

Nitric oxide activates voltage-dependent potassium currents of crustacean skeletal muscle.

Nitric oxide (NO), a radical gas, acts as a multifunctional intra- and intercellular messenger. In the present study we investigated the effects of NO on muscle membrane potassium currents of isolated single muscle fibers from the marine isopods, Idotea baltica, using two-electrode voltage clamp recording techniques. Voltage-activated potassium currents consist of an outward current with fast activation and inactivation kinetics and a delayed, persistent outward current. Both currents were blocked by extracellular 4-aminopyridine and tetraethylammonium; the currents were not blocked by charybdotoxin or apamin. Application of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) or hydroxylamine increased both the early and the delayed outward current in a dose- and time-dependent manner. PTIO, a NO scavenger, suppressed the effect of SNAP. N-Acetyl-dl-penicillamine, a related control compound which does not liberate NO, had no significant effect on outward currents. Methylene blue, a guanylyl cyclase inhibitor, prevented the increase of the outward current while 8-bromo-cGMP increased the current. Our experiments show that potassium currents of Idotea muscle are increased by NO donors. They suggest that NO by stimulating cGMP production mediates the effects on membrane currents involved in regulation of invertebrate muscle excitability.

Nitric oxide is an excitatory modulator in the rostral ventrolateral medulla in rats.

Nitric oxide is a messenger molecule having various functions in the brain. Previous studies have reported conflicting results for the roles of nitric oxide in the rostral ventrolateral medulla, a major center that regulates sympathetic and cardiovascular activities. We hypothesized that in this region, nitric oxide may have a biphasic effect on cardiovascular activity. Microinjection of a low dose (1 nmol) of a nitric oxide donor sodium nitroprusside or a cyclic GMP agonist 8-bromocyclic GMP into this area increased arterial pressure, whereas injection of a nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester or a soluble guanylate cyclase inhibitor methylene blue decreased arterial pressure. Microinjection of a high dose (100 nmol) of sodium nitroprusside decreased arterial pressure and inhibited spontaneous respiration with concomitant production of peroxynitrite, a strong cytotoxic oxidant. Increases in arterial pressure caused by microinjection of L-glutamate were inhibited after preinjection of Nomega-nitro-L-arginine methyl ester or methylene blue. Increases in arterial pressure caused by microinjection of sodium nitroprusside (1 nmol) were inhibited after preinjection of a glutamate receptor antagonist kynurenate. These results suggest that low doses of nitric oxide may increase arterial pressure, whereas high doses of nitric oxide may decrease arterial pressure through cytotoxic effects in the rostral ventrolateral medulla. They also indicate that nitric oxide may stimulate neurons both through activation of the nitric oxide cyclic GMP pathway and through modulation of glutamate receptor stimulation, and therefore, increase arterial pressure in rats.

Cross talk of shear-induced production of prostacyclin and nitric oxide in endothelial cells.

We tested the hypothesis that vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO). Confluent human umbilical vein endothelial cells (HUVEC) were exposed to fluid shear stress at 15 dyn/cm(2) using a cone-plate device, and the concentrations of 6-keto-PGF(1alpha) and NO metabolites (nitrate and nitrite) in the medium were measured with radioimmunoassay and the Greiss method, respectively. Compared with static control, shear stress increased cumulative prostacyclin production by twofold after 90 min of exposure. Inhibition of NO synthase enhanced flow-induced prostacyclin production by twofold without affecting the baseline production. Guanylyl cyclase inhibitor enhanced flow-induced prostacyclin production to the same degree. In contrast, a stable agonist of cGMP attenuated the rapid early phase of flow-dependent prostacyclin production. Shear-induced NO metabolite production was unaffected even after indomethacin inhibited prostacyclin production. We conclude that NO shows an inhibitory effect on prostacyclin production under shear stress and that vessel homeostasis may be maintained through an increase in prostacyclin production when NO synthesis is impaired in endothelial cells.