Nitric oxide-cGMP-mediated vasoconstriction and effects of acetylcholine in the branchial circulation of the eel

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.     

Free hemoglobin induction of pulmonary vascular disease: evidence for an inflammatory mechanism

Cell-free hemoglobin (Hb) exposure may be a pathogenic mediator in the development of pulmonary arterial hypertension (PAH), and when combined with chronic hypoxia the potential for exacerbation of PAH and vascular remodeling is likely more pronounced. We hypothesized that Hb may contribute to hypoxia-driven PAH collectively as a prooxidant, inflammatory, and nitric oxide (NO) scavenger. Using programmable micropump technology, we exposed male Sprague-Dawley rats housed under room air or hypoxia to 12 or 30 mg per day Hb for 3, 5, and 7 wk. Blood pressure, cardiac output, right ventricular hypertrophy, and indexes of pulmonary vascular remodeling were evaluated. Additionally, markers of oxidative stress, NO bioavailability and inflammation were determined. Hb increased pulmonary arterial (PA) pressure, pulmonary vessel wall stiffening, and right heart hypertrophy with temporal and dose dependence in both room air and hypoxic cohorts. Hb induced a modest increase in plasma oxidative stress markers (malondialdehyde and 4-hydroxynonenal), no change in NO bioavailability, and increased lung ICAM protein expression. Treatment with the antioxidant Tempol attenuated Hb-induced pulmonary arterial wall thickening, but not PA pressures or ICAM expression. Chronic exposure to low plasma Hb concentrations (range = 3-10 μM) lasting up to 7 wk in rodents induces pulmonary vascular disease via inflammation and to a lesser extent by Hb-mediated oxidation. Tempol demonstrated a modest effect on the attenuation of Hb-induced pulmonary vascular disease. NO bioavailability was found to be of minimal importance in this model.     

Nitric oxide synthase and cGMP-mediated stimulation of renin secretion

The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney.     

Mechanism of cGMP contribution to the vasodilator response to NO in rat middle cerebral arteries

This study examined the mechanism by which cGMP contributes to the vasodilator response to nitric oxide (NO) in rat middle cerebral arteries (MCA). Administration of a NO donor, diethylaminodiazen-1-ium-1,2-dioate (DEA-NONOate), or 8-bromo-cGMP (8-BrcGMP) increased the diameter of serotonin-preconstricted MCA by 79 +/- 3%. The response to DEA-NONOate, but not 8-BrcGMP, was attenuated by iberiotoxin (10(-7) M) or a 80 mM high-K(+) media, suggesting that activation of K(+) channels contributes to the vasodilator response to NO but not 8-BrcGMP. The effects of NO and cGMP on the vasoconstrictor response to Ca(2+) were also studied in MCA that were permeabilized with alpha-toxin and ionomycin. Elevations in bath Ca(2+) from 10(-8) to 10(-5) M decreased the diameter of permeabilized MCA by 76 +/- 5%. DEA-NONOate (10(-6) M) and 8-BrcGMP (10(-4) M) blunted this response by 60%. Inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (10(-5) M) blocked the inhibitory effect of the NO donor, but not 8-BrcGMP, on Ca(2+)-induced vasoconstriction. 8-BrcGMP (10(-4) M) had no effect on intracellular Ca(2+) concentration ([Ca(2+)](i)) in control, serotonin-stimulated, or alpha-toxin- and ionomycin-permeabilized vascular smooth muscle cells isolated from the MCA. These results indicate that the vasodilator response to NO in rat MCA is mediated by activation of Ca(2+)-activated K(+) channels via a cGMP-independent pathway and that cGMP also contributes to the vasodilator response to NO by decreasing the contractile response to elevations in [Ca(2+)](i).     

Effects of cGMP and sodium nitroprusside on odor responses in turtle olfactory sensory neurons

The effects ofcGMP and sodium nitroprusside (SNP) on odor responses in isolatedturtle olfactory neurons were examined. The inward current induced bydialysis of a mixture of 1 mM cAMP and 1 mM cGMP was similar to thatinduced by dialysis of 1 mM cAMP or 1 mM cGMP alone. After the neuronswere desensitized by the application of 1 mM cGMP, 3 mM8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, did notelicit any current, indicating that both cAMP and cGMP activated thesame channel. Extracellular application of SNP, a nitric oxide (NO)donor, evoked inward currents in a dose-dependent manner. However,application of SNP did not induce any currents after desensitization ofthe cGMP-induced currents, suggesting that SNP-induced currents aremediated via the cGMP-dependent pathway. Application of thecAMP-producing odorants to the neurons induced a large inward currenteven after neurons were desensitized to a high concentration of cGMP orSNP. These results suggest that the transduction pathway independent ofcAMP, cGMP, and NO also contributes to the generation of odor responsesin addition to the cAMP-dependent pathway.

     

The effect of peroxynitrite on the catalytic activity of soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key enzyme of the *NO/cGMP pathway. Many cardiovascular disorders are associated with reduced *NO-mediated effects, while vascular superoxide (O(2)*(-)) production is increased. Both radicals rapidly react to peroxynitrite. We investigated whether peroxynitrite affects the activity and protein expression of sGC in intact vascular preparations. Catalytic sGC activity and expression of the sGC-beta(1) subunit was measured by conversion of radiolabeled GTP and western blot, respectively, using cytosolic extracts from rat aorta that had been incubated for 4 h with *NO/O(2)*(-) systems (devoid of free *NO) generating either 0.13 microM or 7.5 microM peroxynitrite/min. Incubation of rat aorta with 0.13 microM peroxynitrite/min had no effect. In striking contrast, incubation with 7.5 microM peroxynitrite/min resulted in a shift of the concentration-response curve obtained with a *NO donor (p =.0004) and a reduction of maximal specific activity from 3579 +/- 495 to 2422 +/- 265 pmol cGMP/mg/min (p =.036). The expression of the sGC-beta(1) subunit was unchanged. Exposure of aorta to the O(2)*(-) component had no effect, while exposure to the *NO-component reduced sGC expression to 58.8 +/- 7% (p <.001) and maximal sGC activity from 4041 +/- 992 to 1429 +/- 491 pmol cGMP/mg/min (p =.031). These data suggest that continuous generation of extracellular peroxynitrite might interfere with the *NO/cGMP signaling in vascular cells.     

Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism

Exogenous nitric oxide (NO) triggers a preconditioning-like effect in heart via a pathway that is dependent on reactive oxygen species. This study examined the signaling pathway by which the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 2 microM) triggers its anti-infarct effect. Isolated rabbit hearts experienced 30 min of regional ischemia and 120 min of subsequent reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. Infarct size was reduced from 30.5 +/- 3.0% of the risk zone in control hearts to 10.2 +/- 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (2 microM) or the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (200 microM) completely blocked the infarct-sparing effect of SNAP (34.3 +/- 3.8 and 32.2 +/- 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (10 microM), which is a cell-permeable cGMP analog that activates protein kinase G, mimicked the preconditioning effect of SNAP by reducing infarct size to 7.5 +/- 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) or 5-hydroxydecanoate (100 microM; 28.9 +/- 2.7 and 33.6 +/- 5.0% infarction of the risk zone, respectively). To confirm these functional data and the effect of SNAP on the guanylyl cyclase-protein kinase G signaling pathway, cGMP levels were measured. SNAP increased the level from 0.18 +/- 0.04 to 0.61 +/- 0.14 pmol/mg of protein (P < 0.05). These data suggest that exogenous NO triggers the preconditioning effect by initiating a cascade of events including stimulation of guanylyl cyclase to make cGMP, activation of protein kinase G, opening of mitoK(ATP) channels, and, finally, production of reactive oxygen species.     

Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways

We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.     

Estrogen decreases the sensitivity of anterior pituitary to the inhibitory effect of nitric oxide on prolactin release

OBJECTIVE: We investigated the effect of chronic estrogen treatment on the inhibitory action of nitric oxide (NO) on prolactin release. METHODS: The effect of NO on prolactin release was studied in anterior pituitaries of female Wistar rats, intact at random stages, ovariectomized (OVX), and OVX treated for 15 days with 17beta-estradiol (OVX-E(2)). RESULTS: Sodium nitroprusside (NP, 0.5 mM), a NO donor, inhibited prolactin release from anterior pituitaries and was able to stimulate cGMP synthesis in intact and OVX rats. Only a high, supraphysiological concentration of NP (2 mM) inhibited prolactin release from anterior pituitaries of OVX-E(2) rats and increased cGMP synthesis in OVX-E(2) rats. 8-Br-cGMP, a cGMP analogue, decreased prolactin release from anterior pituitaries of OVX rats but did not affect it in OVX-E(2) rats. CONCLUSION: Our results suggest that estrogen may modify the sensitivity of the anterior pituitary to the inhibitory effect of NO on prolactin release by affecting guanylyl cyclase activity and the cGMP pathway.     

Hemodynamic response and oxygen transport in pigs resuscitated with maleimide-polyethylene glycol-modified hemoglobin (MP4).

Cell-free Hb increases systemic and pulmonary pressure and resistance and reduces cardiac output and heart rate in animals and humans, effects that have limited their clinical development as "blood substitutes." The primary aim of this study was to evaluate the hemodynamic response to infusion of several formulations of a new polyethylene glycol (PEG)-modified human Hb [maleimide PEG Hb (MalPEGHb)] in swine, an animal known to be sensitive to Hb-induced vasoconstriction. Anesthetized animals underwent controlled hemorrhage (50% of blood volume), followed by resuscitation (70% of shed volume) with 10% pentastarch (PS), 4% MalPEG-Hb in lactated Ringer (MP4), 4% MalPEG-Hb in pentastarch (HS4), 2% MalPEG-Hb in pentastarch (HS2), or 4% stroma-free Hb in lactated Ringer solution (SFH). Compared with baseline, restoration of blood volume after resuscitation was similar and not significantly different for the PS (103%), HS2 (99%), HS4 (106%), and MP4 (87%) animals but significantly less for the SFH animals (66%) (P < 0.05). All solutions that contained MalPEG-Hb restored mean arterial and pulmonary pressure and cardiac output. Systemic vascular resistance was unchanged, and pulmonary arterial pressure and resistance were increased slightly. Both systemic and pulmonary vascular resistance increased significantly in animals that received SFH, despite less adequate blood volume restoration. Oxygen consumption was maintained in all animals that received MalPEG-Hb, but not PS. Base excess improved only with MalPEG-Hb and PS, but not SFH. Red blood cell O2 extraction was significantly increased in animals that received Hb, regardless of formulation. These data demonstrate resuscitation with MalPEG-human Hb without increasing systemic vascular resistance and support our previous observations in animals suggesting that the efficacy of low concentrations of PEG-Hb in the plasma results from reduced vasoconstriction.     

Sequential activation of soluble guanylate cyclase, protein kinase G and cGMP-degrading phosphodiesterase is necessary for proper induction of long-term potentiation in CA1 of hippocampus. Alterations in hyperammonemia

Long-term potentiation (LTP) is a long-lasting enhancement of synaptic transmission efficacy and is considered the base for some forms of learning and memory. Nitric oxide (NO)-induced formation of cGMP is involved in hippocampal LTP. We have studied in hippocampal slices the effects of application of a tetanus to induce LTP on cGMP metabolism and the mechanisms by which cGMP modulates LTP. Tetanus application induced a transient rise in cGMP, reaching a maximum at 10s and decreasing below basal levels 5 min after the tetanus, remaining below basal levels after 60 min. Soluble guanylate cyclase (sGC) activity increased 5 min after tetanus and returned to basal levels at 60 min. The decrease in cGMP was due to sustained tetanus-induced increase in cGMP-degrading phosphodiesterase activity, which remained activated 60 min after tetanus. Tetanus-induced activation of PDE and decrease of cGMP were prevented by inhibiting protein kinase G (PKG). This indicates that the initial increase in cGMP activates PKG that phosphorylates (and activates) cGMP-degrading PDE, which, in turn, degrades cGMP. Inhibition of sGC, of PKG or of cGMP-degrading phosphodiesterase impairs LTP, indicating that proper induction of LTP involves transient activation of sGC and increase in cGMP, followed by activation of cGMP-dependent protein kinase, which, in turn, activates cGMP-degrading phosphodiesterase, resulting in long-lasting reduction of cGMP content. Hyperammonemia is the main responsible for the neurological alterations found in liver disease and hepatic encephalopathy, including impaired intellectual function. Hyperammonemia impairs LTP in hippocampus by altering the modulation of this sGC-PKG-cGMP-degrading PDE pathway. Exposure of hippocampal slices to 1 mM ammonia completely prevents tetanus-induced decrease of cGMP by impairing PKG-mediated activation of cGMP-degrading phosphodiesterase. This impairment is responsible for the loss of the maintenance of LTP in hyperammonemia, and may be also involved in the cognitive impairment in patients with hyperammonemia and hepatic encephalopathy.     

Effects of the RBC membrane and increased perfusate viscosity on hypoxic pulmonary vasoconstriction.

Red blood cells (RBCs) augment hypoxic pulmonary vasoconstriction (HPV) in part by scavenging of nitric oxide (NO) by Hb (Deem S, Swenson ER, Alberts MK, Hedges RG, and Bishop MJ, Am J Respir Crit Care Med 157: 1181-1186, 1998). We studied the contribution of the RBC compartmentalization of Hb to augmentation of HPV and scavenging of NO in isolated perfused rabbit lungs. Lungs were initially perfused with buffer; HPV was provoked by a 5-min challenge with hypoxic gas (inspired O(2) fraction 0.05). Expired NO was measured continuously. Addition of free Hb to the perfusate (0.25 mg/ml) resulted in augmentation of HPV and a fall in expired NO that were similar in magnitude to those associated with a hematocrit of 30% (intracellular Hb of 100 mg/ml). Addition of dextran resulted in a blunting of HPV after free Hb but no change in expired NO. Blunting of HPV by dextran was not prevented by NO synthase inhibition with N(omega)-nitro-L-arginine and/or cyclooxygenase inhibition. RBC ghosts had a mild inhibitory effect on HPV but caused a small reduction in expired NO. In conclusion, the RBC membrane provides a barrier to NO scavenging and augmentation of HPV by Hb. Increased perfusate viscosity inhibits HPV by an undetermined mechanism.     

Nitric oxide (NO) serves as a retrograde messenger to activate neuronal NO synthase in the spinal cord via NMDA receptors.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.     

Cyclic GMP is a second messenger by which nitric oxide inhibits diaphragm contraction

We have shown that endogenous nitrogen oxides (NOx) modulate excitation-contraction coupling in diaphragm. Because cyclic GMP (cGMP) is a second messenger for nitric oxide (NO) inhibition of smooth muscle contraction, we rested the hypothesis that NO acts via cGMP in diaphragm. Fiber bundles from rat diaphragm were studied in vitro. Immunohistochemical analysis using a cGMP-specific monoclonal antibody confirmed the presence of cGMP in the subsarcolemmal region, near nitric oxide synthase (NOS). cGMP measured by ELISA in control muscle (0.27 pmol/mg +/- 0.01 SE) was significantly increased by the NO donor S-nitroso-N-acetylcysteine 1 mM (0.55+/-0.05; N = 6; P < 0.001). Contractile studies showed that the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) 10 mM increased submaximal (40 Hz) tetanic force (P < 0.0001). L-NNA effects were exaggerated by the guanylate cyclase inhibitor LY83583 5-10 microM; force at 40 Hz was increased (P < 0.001). L-NNA effects were partially reversed by 8-bromo-cGMP 1 mM (8-Br-GMP; a cell-permeable cGMP analogue; P < 0.0001) or dipyridamole 10 microM (DPM; a phosphodiesterase inhibitor; P < 0.0001). 8-Br-GMP and DPM produced more-complete L-NNA reversal in combination (P < 0.0001). We conclude that cGMP functions as a second messenger by which NO inhibits diaphragm contraction.     

Effect of Glutamine Synthesis Inhibition with Methionine Sulfoximine on the Nitric Oxide–Cyclic GMP Pathway in the Rat Striatum Treated Acutely with Ammonia: A Microdialysis Study

Ammonia neurotoxicity is associated with overactivation of N-methyl-d-aspartate (NMDA) receptors leading to enhanced nitric oxide and cyclic GMP synthesis and to accumulation of reactive oxygen and nitrogen species. Ammonia is detoxified in the brain via synthesis of glutamine, which if accumulated in excess contributes to astrocytic swelling, mitochondrial dysfunction and cerebral edema. This study was aimed at testing the hypothesis that the activity of the NMDA/NO/cGMP pathway is controlled by the ammonia-induced production of Gln in the brain. Ammonium chloride (final concentration 5 mM), infused for 40 min to the rat striatum via a microdialysis probe, caused a significant increase in Gln (by 40%), NO oxidation products (nitrite+nitrate = NOx) (by 35%) and cGMP (by 50%) concentration in the microdialysate. A Gln synthetase inhibitor, methionine sulfoximine (MSO, 5 mM), added directly to the microdialysate, completely prevented ammonia-mediated production of Gln, and paradoxically, it increased ammonia-mediated production of NOx and cGMP by 230% and 250%, respectively. Of note, MSO given alone significantly reduced basal Gln concentration in the rat striatum, had no effect on the basal NOx concentration, and attenuated basal concentration of cGMP in the microdialysate by 50%. The results of the present study suggest that Gln, at physiological concentrations, may ameliorate excessive activation of the NO–cGMP pathway by neurotoxic concentrations of ammonia. However, in view of potential direct interference of MSO with the pathway, exogenously added Gln and less toxic modulators of Gln content and/or transport will have to be employed in further studies on the underlying mechanisms. Special issue article in honor of Dr. Frode Fonnum. Wojciech Hilgier and Michal Węgrzynowicz contributed equally to this work.     

Polyethylene glycol and a novel developed polyethylene glycol-nitric oxide normalize arteriolar response and oxidative stress in ischemia-reperfusion

Polyethylene glycol (PEG) has been shown to repair cell membranes and, thus, inhibit free radical production in in vitro and in vivo models. We hypothesized that PEG and newly developed organic nitrate forms of PEG (PEG-NO) could repair endothelial dysfunction in ischemia-reperfusion (I/R) injury in the hamster cheek pouch visualized by intravital fluorescent microscopy. After treatments, we evaluated diameter and RBC velocity and flow in arterioles, as well as lipid peroxides in the systemic blood, perfused capillary length, vascular permeability, leukocyte adhesion, and amount of von Willebrand factor (vWF) in the blood after I/R injury. A control group was treated with 5,000- or 10,000-Da PEG, and three groups were treated with PG1 (1 NO molecule covalently bound to PEG, 5,170 Da), PG8 (8 NO molecules covalently bound to PEG, 11,860 Da), and PG16 (16 NO molecules covalently bound to PEG, 14,060 Da). All animals received 0.5 mg/0.5 ml. Lipid peroxides increased at 5 and 15 min of reperfusion, whereas diameter, RBC velocity, and blood flow decreased in arterioles after I/R injury. Vascular permeability, leukocyte adhesion, and vWF increased significantly. PEG and PG1 attenuated lipid peroxides and vasoconstriction during reperfusion and decreased leukocyte adhesion and vascular permeability. PG8 maintained lipid peroxides at normal levels, increased arteriolar diameter, flow, and perfused capillary length, and decreased vWF level and leukocyte adhesion (P < 0.05). PG16 was less effective than PG1 and PG8. In conclusion, PEG-NO shows promise as a compound that protects microvascular perfusion by normalizing the balance between NO level and excessive production of free radicals in endothelial cells during I/R injury.     

A cell-based nitric oxide reporter assay useful for the identification and characterization of modulators of the nitric oxide/guanosine 3',5'-cyclic monophosphate pathway

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.     

Comparison of the hemodynamic effects of nitric oxide and endothelium-dependent vasodilators in intact lungs

The effects of endothelium-dependent vasodilation on pulmonary vascular hemodynamics were evaluated in a variety of in vivo and in vitro models to determine 1) the comparability of the hemodynamic effects of acetylcholine (ACh), bradykinin (BK), nitric oxide (NO), and 8-bromo-guanosine 3',5'-cyclic monophosphate (cGMP), 2) whether methylene blue is a useful inhibitor of endothelium-dependent relaxing factor (EDRF) activity in vivo, and 3) the effect of monocrotaline-induced pulmonary hypertension on the responsiveness of the pulmonary vasculature to ACh. In isolated rat lungs, which were preconstricted with hypoxia, ACh, BK, NO, and 8-bromo-cGMP caused pulmonary vasodilation, which was not inhibited by maximum tolerable doses of methylene blue. Methylene blue did not inhibit EDRF activity in any model, despite causing increased pulmonary vascular tone and responsiveness to various constrictor agents. There were significant differences in the hemodynamic characteristics of ACh, BK, and NO. In the isolated lung, BK and NO caused transient decreases of hypoxic vasoconstriction, whereas ACh caused more prolonged vasodilation. Pretreatment of these lungs with NO did not significantly inhibit ACh-induced vasodilation but caused BK to produce vasoconstriction. Tachyphylaxis, which was agonist specific, developed with repeated administration of ACh or BK but not NO. Tachyphylaxis probably resulted from inhibition of the endothelium-dependent vasodilation pathway proximal to NO synthesis, because it could be overcome by exogenous NO. Pretreatment with 8-bromo-cGMP decreased hypoxic pulmonary vasoconstriction and, even when the hypoxic pressor response had largely recovered, subsequent doses of ACh and NO failed to cause vasodilation, although BK produced vasoconstriction. These findings are compatible with the existence of feedback inhibition of the endothelium-dependent relaxation by elevation of cGMP levels. Responsiveness to ACh was retained in lungs with severe monocrotaline-induced pulmonary hypertension. Many of these findings would not have been predicted based on in vitro studies and illustrate the importance for expanding studies of EDRF to in vivo and ex vivo models.     

Effect of JBP485 on obstructive jaundice is related to regulation of renal Oat1, Oat3 and Mrp2 expression in ANIT-treated rats

Pulmonary vascular endothelial nitric oxide (NO) synthase (eNOS)-derived NO is the major stimulant of cyclic guanosine 5'-monophosphate (cGMP) production and NO/cGMP-dependent vasorelaxation in the pulmonary circulation. We recently synthesized multiple peptides and reported that an eleven amino acid (SSWRRKRKESS) peptide (P1) but not scrambled P1 stimulated the catalytic activity but not expression of eNOS and causes NO/cGMP-dependent sustained vasorelaxation in isolated pulmonary artery (PA) segments and in lung perfusion models. Since cGMP levels can also be elevated by inhibition of phosphodiesterase type 5 (PDE-5), this study was designed to test the hypothesis that P1-mediated vesorelaxation is due to its unique dual action as NO-releasing PDE-5 inhibitor in the pulmonary circulation. Treatment of porcine PA endothelial cells (PAEC) with P1 caused time-dependent increase in intracellular NO release and inhibition of the catalytic activity of cGMP-specific PDE-5 but not PDE-5 protein expression leading to increased levels of cGMP. Acute hypoxia-induced PA vasoconstriction ex vivo and continuous telemetry monitoring of hypoxia (10% oxygen)-induced elevated PA pressure in freely moving rats were significantly restored by administration of P1. Chronic hypoxia (10% oxygen for 4 weeks)-induced alterations in PA perfusion pressure, right ventricular hypertrophy, and vascular remodeling were attenuated by P1 treatment. These results demonstrate the potential therapeutic effects of P1 to prevent and/or arrest the progression of hypoxia-induced PAH via NO/cGMP-dependent modulation of hemodynamic and vascular remodeling in the pulmonary circulation.     

Kinetics of a cellular nitric oxide/cGMP/phosphodiesterase-5 pathway

Rat platelets served as a model to evaluate quantitatively how guanylate cyclase (GC)-coupled nitric oxide (NO) receptors and phosphodiesterases (here phosphodiesterase-5) interact to transduce NO signals in cells. The platelets expressed mRNA only for the alpha(1) and beta(1) GC-coupled receptor subunits. In intact platelets, the potency of NO for elevating cGMP (EC(50) = 10 nm) was lower than in lysed platelets (EC(50) = 1.7 nm). The limiting activities of GC and phosphodiesterase in intact platelets were both very high, being equivalent to about 100 microm/s. With low phosphodiesterase activity (imposed by 100 microm sildenafil), the cGMP response over time was hyperbolic in shape for a range of NO concentrations or GC activities due to GC desensitization. Without a phosphodiesterase inhibitor, NO generated only brief cGMP transients, peaking after 2-5 s but amounting maximally to about 150 microm cGMP. The transients were caused partly by GC desensitization, which varied in rate (half-time up to 3 s) and extent (up to 80%) depending on the NO concentration, and partly by an enhancement of the phosphodiesterase catalytic activity with time, which was deduced to be up to 30-fold and to occur with a half-time of up to 5 s. The results were simulated by a quantitative model, which also explains the varied shapes of cGMP responses to NO found in other cells. Downstream phosphorylation in platelets was detectable within 2 s, and, with continuous exposure (1 min), this pathway could be engaged by subnanomolar NO concentrations (EC(50) = 0.5 nm).