Nitric oxide exerts feedback inhibition on EDHF-induced coronary arteriolar dilation in vivo

We tested the hypothesis that nitric oxide (NO) inhibits endothelium-derived hyperpolarizing factor (EDHF)-induced vasodilation via a negative feedback pathway in the coronary microcirculation. Coronary microvascular diameters were measured using stroboscopic fluorescence microangiography. Bradykinin (BK)-induced dilation was mediated by EDHF, when NO and prostaglandin syntheses were inhibited, or by NO when EDHF and prostaglandin syntheses were blocked. Specifically, BK (20, 50, and 100 ng. kg(-1). min(-1) ic) caused dose-dependent vasodilation similarly before and after administration of N(G)-monomethyl-L-arginine (L-NMMA) (3 micromol/min ic for 10 min) and indomethacin (Indo, 10 mg/kg iv). The residual dilation to BK with L-NMMA and Indo was completely abolished by suffusion of miconazole or an isosmotic buffer containing high KCl (60 mM), suggesting that this arteriolar vasodilation is mediated by the cytochrome P-450 derivative EDHF. BK-induced dilation was reduced by 39% after inhibition of EDHF and prostaglandin synthesis, and dilation was further inhibited by combined blockade with L-NMMA to a 74% reduction in the response. This suggests an involvement for NO in the vasodilation. After dilation to BK was assessed with L-NMMA and Indo, sodium nitroprusside (SNP, 1-3 microgram. kg(-1). min(-1) ic), an exogenous NO donor, was administered in a dose to increase the diameter to the original control value. Dilation to BK was virtually abolished when administered concomitantly with SNP during L-NMMA and Indo (P < 0.01 vs. before SNP), suggesting that NO inhibits EDHF-induced dilation. SNP did not affect adenosine- or papaverine-induced arteriolar dilation in the presence of L-NMMA and Indo, demonstrating that the effect of SNP was not nonspecific. In conclusion, our data are the first in vivo evidence to suggest that NO inhibits the production and/or action of EDHF in the coronary microcirculation.     

Skeletal muscle blood flow and oxygen uptake at rest and during exercise in humans: a pet study with nitric oxide and cyclooxygenase inhibition

The aim of the present study was to determine the effect of nitric oxide and prostanoids on microcirculation and oxygen uptake, specifically in the active skeletal muscle by use of positron emission tomography (PET). Healthy males performed three 5-min bouts of light knee-extensor exercise. Skeletal muscle blood flow and oxygen uptake were measured at rest and during the exercise using PET with H(2)O(15) and (15)O(2) during: 1) control conditions; 2) nitric oxide synthase (NOS) inhibition by arterial infusion of N(G)-monomethyl-L-arginine (L-NMMA), and 3) combined NOS and cyclooxygenase (COX) inhibition by arterial infusion of L-NMMA and indomethacin. At rest, inhibition of NOS alone and in combination with indomethacin reduced (P < 0.05) muscle blood flow. NOS inhibition increased (P < 0.05) limb oxygen extraction fraction (OEF) more than the reduction in muscle blood flow, resulting in an ～20% increase (P < 0.05) in resting muscle oxygen consumption. During exercise, muscle blood flow and oxygen uptake were not altered with NOS inhibition, whereas muscle OEF was increased (P < 0.05). NOS and COX inhibition reduced (P < 0.05) blood flow in working quadriceps femoris muscle by 13%, whereas muscle OEF and oxygen uptake were enhanced by 51 and 30%, respectively. In conclusion, by specifically measuring blood flow and oxygen uptake by the use of PET instead of whole limb measurements, the present study shows for the first time in humans that inhibition of NO formation enhances resting muscle oxygen uptake and that combined inhibition of NOS and COX during exercise increases muscle oxygen uptake.     

Functional evidence for nitric oxide production by skeletal-muscle mitochondria from lipopolysaccharide-treated mice

The possible existence of a mitochondrially localized nitric oxide (NO) synthase (mtNOS) is controversial. To clarify this, we studied the ability of intact mitochondria to generate NO and the effect of mitochondrial NO on respiration. Respiratory rates and oxygen kinetics (P(50) values) were determined by high-resolution respirometry in skeletal-muscle mitochondria from control mice and mice injected with Escherichia coli lipopolysaccharide (LPS). In the presence of the NOS substrate L-arginine, mitochondria from LPS-treated mice had lower respiration rates and higher P(50) values than control animals. These effects were prevented by the NOS inhibitor L-NMMA. Our results suggest that mitochondrially derived NO is generated by an LPS-inducible NOS protein other than iNOS and modulates oxygen consumption in mouse skeletal muscle.     

Nitric oxide and prostaglandin systems in the stimulation of hypothalamic-pituitary-adrenal axis by neurotransmitters and neurohormones.

The review presents our results on the regulatory role of prostaglandins (PG) and nitric oxide (NO) in the activation of hypothalamic-pituitary-adrenal (HPA) axis by cholinergic, adrenergic and histaminergic systems and by neurohormones: corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) under basal conditions. The synthesis of endogenous PG or NO was inhibited by non-selective and selective cyclooxygenase (COX) antagonists and nitric oxide synthase (NOS) blockers given 15 min before the respective receptor agonist and HPA axis activity was assessed 1 h later by measuring plasma ACTH and serum corticosterone levels. The muscarinic agent - carbachol-induced HPA response was considerably supressed by piroxicam, a predominantly constitutive cyclooxygenase (COX-1) inhibitor and significantly diminished by indomethacin, a non-selective COX blocker, but was unaffected by compound NS-398, an inducible cyclooxygenase (COX-2) antagonist. A non-selective NOS antagonist L-NAME and neuronal NOS blocker L-NNA significantly intensified the carbachol-induced corticosterone secretion. The nicotine-induced increase in ACTH and corticosterone response was significantly supressed by piroxicam, and diminished by indomethacin, but was significantly augmented by L-NAME and L-NNA. The inhibition of PG synthesis by indomethacin totally abolished or reversed the increase of nicotine-induced hormone responses to both NOS blockers. The i.c.v. phenylephrine, an alpha(1)-adrenergic receptor agonist - evoked HPA response was significantly impaired by piroxicam and compound NS-398 and more potently reduced by L-NAME. The i.c.v. clonidine, an alpha(2)-adrenergic agonist - elicited HPA response was also considerably decreased by piroxicam, compound NS-398 and L-NAME. By contrast, the stimulatory effect of i.c.v. isoprenaline, a non-selective beta-adrenergic agonist, was not altered by either COX or NOS inhibitors. The i.c.v. histamine- and HTMT, a histamine H(1)-agonist-induced ACTH and corticosterone response were significantly diminished by piroxicam and indomethacin, respectively. Compound NS-398, did not markedly alter the HPA response to HTMT or amthamine, a histamine H(2) receptor agonist. Inhibition of endogenous NO synthesis by a neuronal NOS inhibitor 7-nitroindazole markedly enhanced the histamine-induced hormone secretion, abolished the HTMT-induced response and did not substantially alter the amthamine-evoked ACTH and corticosterone secretion. COX blockers did not significantly affect the CRH-induced HPA response and the inhibition of NO synthesis by L-NNA markedly intensified ACTH response. The vasopressin-stimulated increase in HPA response, was considerably reduced by the inhibition of PG synthesis by both COX antagonists while inhibition of NO synthesis by NOS blockers greatly enhanced this response. The involvement of PG and NO in the neurohormonal regulation of HPA activity depends mainly on greatly complex and tightly regulated mechanisms at the level of second messengers IP(3) and adenylyl cyclase systems.     

Role of prostanoids and nitric oxide inhibition in rats with experimental hepatic fibrosis

Nitric oxide (NO) and prostaglandins have been proposed as vasodilator substances involved in peripheral vasodilatation characteristic of the liver cirrhosis. A link between NO and prostanoids has been suggested. The present study investigated the effect of simultaneous blockade of both, NO synthase (NOS) and cyclooxigenase (COX) in sham-operated (SO), or rats with bile-duct ligation (BDL) in the development of liver fibrosis. Animals were distributed in two groups SO (n=15) or BDL (n=15). Treatments (5 days) started three weeks after surgical procedure. Both, SO and BDL animals were treated with indomethacin (INDO) (5 mg/kg/day) alone, with NG-nitro-L-arginine-methyl-ester (NAME) (4 mg/kg/day) alone or with INDO and NAME combination at the same doses. At the end of follow-up body weight, packed cell volume, mean arterial blood pressure (MAP) and heart rate were measured. Liver tissue was processed for histological studies. In this study, BDL animals showed a decreased MAP. Treatment with L-NAME in BDL rats increased MAP. The chronic COX inhibition alone did not play an important role in the haemodynamic changes. The BDL produced a loss of hepatic structure, with ductular metaplasia that occupied the greater part of the hepatic parenchyma. Also, an important degree of fibrosis was observed. Both NO and PG synthesis inhibitors, alone or in combination, induced enhancing collagen fiber deposition in the hepatic parenchyma. These findings support the notion that the interaction between the NOS and COX pathways should be relevant in hepatic cirrhosis in which both NOS and COX are induced.     

Mechanisms of ATP-mediated vasodilation in humans: modest role for nitric oxide and vasodilating prostaglandins

ATP is an endothelium-dependent vasodilator, and findings regarding the underlying signaling mechanisms are equivocal. We sought to determine the independent and interactive roles of nitric oxide (NO) and vasodilating prostaglandins (PGs) in ATP-mediated vasodilation in young, healthy humans and determine whether any potential role was dependent on ATP dose or the timing of inhibition. In protocol 1 (n = 18), a dose-response curve to intrabrachial infusion of ATP was performed before and after both single and combined inhibition of NO synthase [N(G)-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (ketorolac). Forearm blood flow (FBF) was measured via venous occlusion plethysmography and forearm vascular conductance (FVC) was calculated. In this protocol, neither individual nor combined NO/PG inhibition had any effect on the vasodilatory response (P = 0.22-0.99). In protocol 2 (n = 16), we determined whether any possible contribution of both NO and PGs to ATP vasodilation was greater at low vs. high doses of ATP and whether inhibition during steady-state infusion of the respective dose of ATP impacted the dilation. FBF in this protocol was measured via Doppler ultrasound. In protocol 2, infusion of low (n = 8)- and high-dose (n = 8) ATP for 5 min evoked a significant increase in FVC above baseline (low = 198 ± 24%; high = 706 ± 79%). Infusion of L-NMMA and ketorolac together reduced steady-state FVC during both low- and high-dose ATP (P < 0.05), and in a subsequent trial with continuous NO/PG blockade, the vasodilator response from baseline to 5 min of steady-state infusion was similarly reduced for both low (ΔFVC = -31 ± 11%)- and high-dose ATP (ΔFVC -25 ± 11%; P = 0.70 low vs. high dose). Collectively, our findings indicate a potential modest role for NO and PGs in the vasodilatory response to exogenous ATP in the human forearm that does not appear to be dose or timing dependent; however, this is dependent on the method for assessing forearm vascular responses. Importantly, the majority of ATP-mediated vasodilation is independent of these putative endothelium-dependent pathways in humans.     

Cardioprotective role of endogenous hydrogen peroxide during ischemia-reperfusion injury in canine coronary microcirculation in vivo

We have recently demonstrated that endogenous H2O2 plays an important role in coronary autoregulation in vivo. However, the role of H2O2 during coronary ischemia-reperfusion (I/R) injury remains to be examined. In this study, we examined whether endogenous H2O2 also plays a protective role in coronary I/R injury in dogs in vivo. Canine subepicardial small coronary arteries (>or=100 microm) and arterioles (<100 microm) were continuously observed by an intravital microscope during coronary I/R (90/60 min) under cyclooxygenase blockade (n=50). Coronary vascular responses to endothelium-dependent vasodilators (ACh) were examined before and after I/R under the following seven conditions: control, nitric oxide (NO) synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA), catalase (a decomposer of H2O2), 8-sulfophenyltheophylline (8-SPT, an adenosine receptor blocker), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA, an inhibitor of large-conductance Ca2+-sensitive potassium channels), and L-NMMA+catalase+8-SPT. Coronary I/R significantly impaired the coronary vasodilatation to ACh in both sized arteries (both P<0.01); L-NMMA reduced the small arterial vasodilatation (both P<0.01), whereas it increased (P<0.05) the ACh-induced coronary arteriolar vasodilatation associated with fluorescent H2O2 production after I/R. Catalase increased the small arterial vasodilatation (P<0.01) associated with fluorescent NO production and increased endothelial NOS expression, whereas it decreased the arteriolar response after I/R (P<0.01). L-NMMA+catalase, L-NMMA+TEA, or L-NMMA+catalase+8-SPT further decreased the coronary vasodilatation in both sized arteries (both, P<0.01). L-NMMA+catalase, L-NMMA+TEA, and L-NMMA+catalase+8-SPT significantly increased myocardial infarct area compared with the other four groups (control, L-NMMA, catalase, and 8-SPT; all, P<0.01). These results indicate that endogenous H2O2, in cooperation with NO, plays an important cardioprotective role in coronary I/R injury in vivo.     

In vitro pharmacological actions of sinomenine on the smooth muscle and the endothelial cell activity in rat aorta

Vasodilating actions of sinomenine were examined using rat aorta ring strips. Sinomenine (0.1 to 100 microM) dilated norepinephrine (NE, 5 microM)-induced vasoconstriction in a concentration-dependent manner reaching 68.8+/-5.1% (n=6, P<0.01) at a concentration of 100 microM. Sinomenine (100 microM) also attenuated KCl (60 mM) and phorbol 12, 13-dibutyrate (PDB, a protein kinase C, PK-C, activator, 300 nM)-induced vasoconstriction by 86.9+/-8.5% (n=6, P<0.01) and 49.9+/-9.8% (n=6, P<0.01), respectively. Pretreatment with nicardipine (a Ca2+ channel antagonist), staurosporine (a PK-C inhibitor), NG-monomethyl-L-arginine acetate (L-NMMA, a nitric oxide, NO, synthesis inhibitor), and indomethacin (a cyclooxygenase inhibitor) were carried out. Nicardipine (0.1 microM) led to a significant decrease in the vasodilating potential of sinomenine (at 100 microM, 68.8+/-5.1% vs. 35.5+/-6.9%; n=5, P<0.001). Pretreatment with staurosporine (30 nM) reduced sinomenine-associated vasodilation from 68.8+/-5.1% to 49.5+/-7.7% (n=5, P<0.001), and L-NMMA (100 microM) and indomethacin (10 microM), to 25.3+/-2.3% (n=5, P<0.001) and to 37.1+/-9.3% (n=5, P<0.001), respectively. The responses were almost similar to the results without endothelium. Therefore, these results indicate that sinomenine causes the vasorelaxation by the mechanisms involved with the inhibitions of Ca2+ channel and PK-C activity, and also with the activations of NO and prostaglandin (PG) I2 syntheses in endothelium.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Nascent EDHF-mediated cerebral vasodilation in ovariectomized rats is not induced by eNOS dysfunction

In estrogen-depleted [i.e., ovariectomized (Ovx)] animals, an endothelium-derived hyperpolarizing factor (EDHF)-like mechanism may arise to, at least partially, replace endothelial nitric oxide (NO) synthase (eNOS)-derived NO in modulating cerebral arteriolar tone. Additional findings show that eNOS expression and function is restored in estrogen-treated Ovx female rats, while the nascent EDHF-like activity disappears. Because NO has been linked to repression of EDHF activity in the periphery, the current study was undertaken to examine whether the nascent EDHF role in cerebral vessels of Ovx females relates to a chronically repressed eNOS-derived NO-generating function. We compared the effects of chronic NOS inhibition with Nomega-nitro-L-arginine-methyl ester (L-NAME; 100 mg. kg-1. day-1 for 3 wk) on EDHF-mediated pial arteriolar vasodilation in anesthetized intact, Ovx, and 17beta-estradiol-treated (0.1 mg. kg-1. day-1 ip, 1 wk) Ovx (OVE) female rats as well as in male rats that were prepared with closed cranial windows. In the chronic NOS inhibition groups, pial arteriolar responses were monitored in the absence (all groups) and presence (females only) of indomethacin (Indo; 10 mg/kg iv). Finally, the gap junction inhibitory peptide Gap 27 (300 muM) was applied to block EDHF-related vasodilation. NO donor (S-nitroso-N-acetyl-penicillamine) responses were similar in all rats studied. Acetylcholine (ACh) reactivity was virtually absent in control Ovx rats and chronically NOS-inhibited intact female, OVE, and male rats. However, a partial recovery of ACh reactivity was seen in L-NAME-treated Ovx females. In addition, in the presence of L-NAME, a normal CO2 reactivity was observed in all females, whereas a 50% reduction in CO2 reactivity was seen in males. In intact and OVE rats, both chronic and acute (NG-nitro-L-arginine suffusion) NOS inhibition, combined with Indo, depressed ADP-induced dilation by > or =50%, and subsequent application of Gap 27 had no further effect on ADP-induced vasodilation. ADP reactivity was retained in Ovx rats after combined chronic NOS inhibition and acute Indo, but was attenuated significantly by Gap 27. In males, Gap 27 had no effect on arteriolar reactivity. Taken together, our data demonstrate that in the cerebral microcirculation, NO does not have an inhibitory effect on EDHF production or action. The increased EDHF-like function in chronic estrogen-depleted animals is not due to eNOS deficiency, suggesting a more direct effect of estrogen in modulating EDHF-mediated cerebral vasodilation.     

Relationship between muscle sympathetic nerve activity and systemic hemodynamics during nitric oxide synthase inhibition in humans

Large interindividual differences exist in resting sympathetic nerve activity (SNA) among normotensive humans with similar arterial pressure (AP). We recently showed inverse relationships of resting SNA with cardiac output (CO) and vascular adrenergic responsiveness that appear to balance the influence of differences in SNA on blood pressure. In the present study, we tested whether nitric oxide (NO)-mediated vasodilation has a role in this balance by evaluating hemodynamic responses to systemic NO synthase (NOS) inhibition in individuals with low and high resting muscle SNA (MSNA). We measured MSNA via peroneal microneurography, CO via acetylene uptake and AP directly, at baseline and during increasing systemic doses of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). Baseline MSNA ranged from 9 to 38 bursts/min (13 to 68 bursts/100 heartbeats). L-NMMA caused dose-dependent increases in AP and total peripheral resistance and reflex decreases in CO and MSNA. Increases in AP with L-NMMA were greater in individuals with high baseline MSNA (PANOVA<0.05). For example, after 8.5 mg/kg of L-NMMA, in the low MSNA subgroup (n=6, 28+/-4 bursts/100 heartbeats), AP increased 9+/-1 mmHg, whereas in the high-MSNA subgroup (n=6, 58+/-3 bursts/100 heartbeats), AP increased 15+/-2 mmHg (P<0.01). The high-MSNA subgroup had lower baseline CO and smaller decreases in CO with L-NMMA, but changes in total peripheral resistance were not different between groups. We conclude that differences in CO among individuals with varying sympathetic traffic have important hemodynamic implications during disruption of NO-mediated vasodilation.     

Nitric oxide modulates sympathoexcitatory cardiac-cardiovascular reflexes elicited by bradykinin

A number of studies have demonstrated an important role for nitric oxide (NO) in central and peripheral neural modulation of sympathetic activity. To assess the interaction and integrative effects of NO release and sympathetic reflex actions, we investigated the influence of inhibition of NO on cardiac-cardiovascular reflexes. In anesthetized, sinoaortic-denervated and vagotomized cats, transient reflex increases in arterial blood pressure (BP) were induced by application of bradykinin (BK, 0.1-10 microg/ml) to the epicardial surface of the heart. The nonspecific NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA, 10 mg/kg iv) was then administered and stimulation was repeated. L-NMMA increased baseline mean arterial pressure (MAP) from 129 +/- 8 to 152 +/- 9 mmHg and enhanced the change in MAP in response to BK from 32 +/- 3 to 39 +/- 5 mmHg (n = 9, P < 0.05). Pulse pressure was significantly enhanced during the reflex response from 6 +/- 4 to 27 +/- 6 mmHg after L-NMMA injection due to relatively greater potentiation of the rise in systolic BP. Both the increase in baseline BP and the enhanced pressor reflex were reversed by L-arginine (30 mg/kg iv). Because L-NMMA can inhibit both brain and endothelial NOS, the effects of 7-nitroindazole (7-NI, 25 mg/kg ip), a selective brain NOS inhibitor, on the BK-induced cardiac-cardiovascular pressor reflex also were examined. In contrast to L-NMMA, we observed significant reduction of the pressor response to BK from 37 +/- 5 to 18 +/- 3 mmHg 30 min after the administration of 7-NI (n = 9, P < 0.05), an effect that was reversed by L-arginine (300 mg/kg iv, n = 7). In a vehicle control group for 7-NI (10 ml of peanut oil ip), the pressor response to BK remained unchanged (n = 6, P > 0.05). In conclusion, neuronal NOS facilitates, whereas endothelial NOS modulates, the excitatory cardiovascular reflex elicited by chemical stimulation of sympathetic cardiac afferents.     

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.     

Interactions between angiotensin II and nitric oxide during exercise in normal and heart failure rats.

We hypothesized that nitric oxide (NO) opposes ANG II-induced increases in arterial pressure and reductions in renal, splanchnic, and skeletal muscle vascular conductance during dynamic exercise in normal and heart failure rats. Regional blood flow and vascular conductance were measured during treadmill running before (unblocked exercise) and after 1) ANG II AT(1)-receptor blockade (losartan, 20 mg/kg ia), 2) NO synthase (NOS) inhibition [N(G)-nitro-L-arginine methyl ester (L-NAME); 10 mg/kg ia], or 3) ANG II AT(1)-receptor blockade + NOS inhibition (combined blockade). Renal conductance during unblocked exercise (4.79 +/- 0.31 ml x 100 g(-1) x min(-1) x mmHg(-1)) was increased after ANG II AT(1)-receptor blockade (6.53 +/- 0.51 ml x 100 g(-1) x min(-1) x mmHg(-1)) and decreased by NOS inhibition (2.12 +/- 0.20 ml x 100 g(-1) x min(-1) x mmHg(-1)) and combined inhibition (3.96 +/- 0.57 ml x 100 g(-1) x min(-1) x mmHg(-1); all P < 0.05 vs. unblocked). In heart failure rats, renal conductance during unblocked exercise (5.50 +/- 0.66 ml x 100 g(-1) x min(-1) x mmHg(-1)) was increased by ANG II AT(1)-receptor blockade (8.48 +/- 0.83 ml x 100 g(-1) x min(-1) x mmHg(-1)) and decreased by NOS inhibition (2.68 +/- 0.22 ml x 100 g(-1) x min(-1) x mmHg(-1); both P < 0.05 vs. unblocked), but it was unaltered during combined inhibition (4.65 +/- 0.51 ml x 100 g(-1) x min(-1) x mmHg(-1)). Because our findings during combined blockade could be predicted from the independent actions of NO and ANG II, no interaction was apparent between these two substances in control or heart failure animals. In skeletal muscle, L-NAME-induced reductions in conductance, compared with unblocked exercise (P < 0.05), were abolished during combined inhibition in heart failure but not in control rats. These observations suggest that ANG II causes vasoconstriction in skeletal muscle that is masked by NO-evoked dilation in animals with heart failure. Because reductions in vascular conductance between unblocked exercise and combined inhibition were less than would be predicted from the independent actions of NO and ANG II, an interaction exists between these two substances in heart failure rats. L-NAME-induced increases in arterial pressure during treadmill running were attenuated (P < 0.05) similarly in both groups by combined inhibition. These findings indicate that NO opposes ANG II-induced increases in arterial pressure and in renal and skeletal muscle resistance during dynamic exercise.     

Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na(+)-K(+) pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become "uncoupled" and preferentially synthesize O(2)(.-), which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na(+)-K(+) pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. L-arginine (L-arg; 500 micromol/l) increased DAF-2 DA fluorescence by 51% compared with control (n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na(+)-K(+) pump current (I(p)). Mean I(p) of 0.35 +/- 0.03 pA/pF (n = 44) was increased to 0.48 +/- 0.03 pA/pF (n = 7, P < 0.05) by 10 micromol/l L-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O(2)(.-)-sensitive dye dihydroethidium compared with control (n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured I(p) with 100 micromol/l paraquat included in patch pipette solutions. This decreased I(p) to 0.28 +/- 0.03 pA/pF (n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na(+)-K(+) pump, whereas uncoupling of NOS causes O(2)(.-)-mediated pump inhibition.     

NO signalling in cytokinin-induced programmed cell death

Cell death can be induced by cytokinin 6-benzylaminopurine (BA) at high dosage in suspension-cultured Arabidopsis cells. Herein, we provide evidence that BA induces nitric oxide (NO) synthesis in a dose-dependent manner. A reduction in cell death can be observed when the cytokinin is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or the nitric oxide synthase (NOS) inhibitors: 2-aminoethyl-isothiourea (AET) and N^G.-monomethyl- l -arginine ( l -NMMA), which suggests that NO is produced via a NOS and is a signalling component of this form of programmed cell death. In BA-treated cells, mitochondrial functionality is altered via inhibition of respiration. This inhibition can be prevented by addition of either cPTIO or AET implying that NO acts at the mitochondrial level.     

Prostaglandins mediate suppression of lymphocyte proliferation and cytokine synthesis in acute Trypanosoma cruzi infection

Suppression of host lymphoproliferative responses to mitogens and Ag is characteristically seen during acute infection with the protozoan parasite Trypanosoma cruzi. We investigated the reciprocal regulation of prostaglandins (PG), TNF-alpha, and nitric oxide (NO) production and their effects on cytokine production and lymphoproliferative responses to parasite Ag and to Con A by spleen cells (SC) from T.-cruzi-infected mice. Large amounts of PGE2, TNF-alpha, and NO were produced during infection. TNF-alpha stimulated PG and NO synthesis, while both mediators inhibited TNF-alpha synthesis. Blocking PG also reduced NO synthesis indicating that PG stimulate NO production. Treatment with indomethacin or NMLA stimulated lymphoproliferation on days 6 and 22 of infection; on day 14, when suppression of proliferation and NO production was maximal, combined inhibition of NO and PG production restored parasite Ag specific and Con A proliferative responses. Blocking PG or NO production increased IL-2, IFN-gamma, and TNF-alpha, but not IL-12 production by SC; IL-10 levels were not reduced. Indomethacin-treated infected mice had higher mortality compared to untreated infected animals. The data indicate that PG, together with NO and TNF-alpha, participate in a complex circuit that controls lymphoproliferative and cytokine responses in T. cruzi infection.     

Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase

Nitric oxide (NO) and its derivatives inhibit mitochondrial respiration by a variety of means. Nanomolar concentrations of NO immediately, specifically and reversibly inhibit cytochrome oxidase in competition with oxygen, in isolated cytochrome oxidase, mitochondria, nerve terminals, cultured cells and tissues. Higher concentrations of NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Isolated mitochondria, cultured cells, isolated tissues and animals in vivo display respiratory inhibition by endogenously produced NO from constitutive isoforms of NO synthase (NOS), which may be largely mediated by NO inhibition of cytochrome oxidase. Cultured cells expressing the inducible isoform of NOS (iNOS) can acutely and reversibly inhibit their own cellular respiration and that of co-incubated cells due to NO inhibition of cytochrome oxidase, but after longer-term incubation result in irreversible inhibition of cellular respiration due to NO or its derivatives. Thus the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen.     

The role of endogenous nitric oxide in inhibition of ischemia/reperfusion-induced cardiomyocyte apoptosis

The effect of nitric oxide (NO) synthase inhibition on apoptosis of cardiomyocytes during ischemia/reperfusion was investigated. Isolated perfused guinea-pig hearts were subjected to 35 min ischemia (I) followed by 30 min reperfusion (IR) in the presence or absence of NO synthase inhibitors, L-NAME or L-NMMA or a superoxide scavenger, SOD. Apoptosis was assessed by immunohistochemistry (TUNEL assay, Bax protein staining), by spectrophotometric measurement of cytochrome oxidase activity (COX), and by ultrastructural analysis. Inhibition of NOS significantly increased apoptosis with activation of Bax protein and decrease of COX. SOD infusion had a protective effect on these apoptotic markers. The results suggest that endogenous NO synthesis during I/R protects the heart against apoptotic cell death.     

Nitric oxide and prostaglandins influence local skeletal muscle blood flow during exercise in humans: coupling between local substrate uptake and blood flow

Synergic action of nitric oxide (NO) and prostaglandins (PG) in the regulation of muscle blood flow during exercise has been demonstrated. In the present study, we investigated whether these vasodilators also regulate local blood flow, flow heterogeneity, and glucose uptake within the exercising skeletal muscle. Skeletal muscle blood flow was measured in seven healthy young men using near-infrared spectroscopy and indocyanine green and muscle glucose uptake using positron emission tomography and 2-fluoro-2-deoxy-D-[(18)F]glucose without and with local blockade of NO and PG at rest and during one-legged dynamic knee-extension exercise. Local blockade was produced by infusing nitro-L-arginine methyl ester and indomethacin directly in the muscle via a microdialysis catheter. Blood flow and glucose uptake were measured in the region of blockade and in two additional regions of vastus lateralis muscle 1 and 4 cm away from the infusion of blockers. Local blockade during exercise at 25 and 40 watts significantly decreased blood flow in the infusion region and in the region 1 cm away from the site of infusion but not in the region 4 cm away. During exercise, muscle glucose uptake did not show any regional differences in response to blockade. These results show that NO and PG synergistically contribute to the local regulation of blood flow in skeletal muscle independently of muscle glucose uptake in healthy young men. Thus these vasodilators can play a role in regulating microvascular blood flow in localized regions of vastus lateralis muscle but do not influence regional glucose uptake. The findings suggest that local substrate uptake in skeletal muscle can be regulated independently of regional changes in blood flow.