Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent. A novel action of insulin to increase nitric oxide release

The purpose of this study was to examine whether insulin's effect to vasodilate skeletal muscle vasculature is mediated by endothelium-derived nitric oxide (EDNO). N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO synthase, was administered directly into the femoral artery of normal subjects at a dose of 16 mg/min and leg blood flow (LBF) was measured during an infusion of saline (NS) or during a euglycemic hyperinsulinemic clamp (HIC) designed to approximately double LBF. In response to the intrafemoral artery infusion of L-NMMA, LBF decreased from 0.296 +/- 0.032 to 0.235 +/- 0.022 liters/min during NS and from 0.479 +/- 0.118 to 0.266 +/- 0.052 liters/min during HIC, P < 0.03. The proportion of NO-dependent LBF during NS and HIC was approximately 20% and approximately 40%, respectively, P < 0.003 (NS vs. HIC). To elucidate whether insulin increases EDNO synthesis/release or EDNO action, vasodilative responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator methacholine chloride (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP) were studied in normal subjects during either NS or HIC. LBF increments in response to intrafemoral artery infusions of MCh but not SNP were augmented during HIC versus NS, P < 0.03. In summary, insulin-mediated vasodilation is EDNO dependent. Insulin vasodilation of skeletal muscle vasculature most likely occurs via increasing EDNO synthesis/release. Thus, insulin appears to be a novel modulator of the EDNO system.     

Early posttraumatic increase in production of nitric oxide in humans

In nephropathic cystinosis, corneal cystine crystals cause severe photophobia and corneal erosions. Topical cysteamine dissolves these crystals, but cannot be marketed because it rapidly oxidizes to the disulfide form, cystamine, at room temperature. Since cystamine itself could be used commercially, we compared the efficacy of cystamine and cysteamine with respect to cystine crystal dissolution in a randomized, double-masked clinical trial. One eye each of 14 patients with cystinosis was randomized to either cystamine or cysteamine, 0.5%, with 0.01% benzalkonium chloride; the companion eye was treated with the alternate preparation. Corneal crystals were photographed and a density score was assigned to each slide based on 13 standard slides. After 8-20 months, 6 patients showed significant reduction of the corneal crystal score in only one eye. In each case, the improved eye was the cysteamine-treated eye. Theoretically, cysteamine should dissolve both intracellular and extracellular crystals, whereas cystamine should dissolve only intracellular crystals because it must first be reduced to the free thiol by the cytoplasmic-reducing environment. Hence, the lack of efficacy of the disulfide cystamine suggests that some corneal cystine crystals in cystinosis patients are extracellular, and that another form of stable, topical cysteamine must be developed for cystinosis patients.     

Fibrinogen is negatively correlated with aerobic working capacity in football players

Subjective quality of life in a group of chronic schizophrenia outpatients was assessed within 1 1/2 years (n = 50) and 4-6 weeks (n = 47) using the Berliner Lebensqualit?tsprofil (German version of the Lancashire Quality of Life Profile). There had been no systematic therapeutic interventions meanwhile. Stability and constancy for the longer interval was similar to results in the general population. The statistics for the shorter interval justify in particular use of the profile's total score and general well-being for the measurement of change. In contrast to cross-sectional data and the longer observation period, no significant common variation between psychopathological symptoms and subjective quality of life could be found for the shorter period.     

An increase in intracellular cyclic AMP modulates nitric oxide production in IFN-gamma-treated macrophages

Macrophages treated with IFN-gamma alone are stimulated to produce nitric oxide. The level of nitric oxide production can be enhanced significantly when IFN-gamma treatment is combined with other agents (e.g., LPS, TNF-alpha, IL-2, etc.). We tested the hypothesis that cAMP plays a role in the IFN-gamma-induced activation of macrophages. Our experiments indicate that factors that increase the concentration of cAMP in the murine macrophage cell line ANA-1 can also enhance IFN-gamma-induced production of nitric oxide. PGE2 and cholera toxin increased the production of nitrite (an indicator of nitric oxide production) in IFN-gamma-treated ANA-1 macrophages by at least twofold. These factors produced no increase in nitric oxide production in the absence of IFN-gamma treatment. The increase in nitric oxide production corresponded to an increase in the accumulation of nitric oxide synthase mRNA without a change in stability of mRNA. Dibutyryl cAMP and Sp-cAMPs (a selective activator of cAMP-dependent protein kinase I and II) also increased nitric oxide production in IFN-gamma-treated macrophages. However, at very high concentrations (i.e., >100 microM), the stimulatory effect was decreased. These studies indicate that elevation of intracellular cAMP causes a dose-dependent, biphasic alteration of IFN-gamma-induced nitric oxide production in murine macrophages. Moreover, they suggest that agents that affect nitric oxide synthesis may do so via modulation of the cAMP second messenger system.     

Monocyte-vascular smooth muscle cell interaction enhances nitric oxide production

OBJECTIVE: The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis. We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression. METHODS: NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting. RESULTS: Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha. The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody. CONCLUSIONS: The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.     

Regulation of nitric oxide production in response to skeletal muscle activation

Nitric oxide (NO) is synthesized in normal muscle fibers by the neuronal (nNOS) and the endothelial (ecNOS) isoforms of nitric oxide synthase (NOS). NO contributes to the regulation of several processes such as excitation-contraction coupling and mitochondrial respiration. We assessed in this study whether NO production is regulated in response to an acute increase in muscle activation. Three groups of anesthetized, tracheostomized, spontaneously breathing rats were examined after an experimental period of 3 h. Group 1 served as a control (no loading), whereas groups 2 and 3 were exposed to moderate and severe inspiratory resistive loads, respectively, which elicited tracheal pressures of 30 and 70% of maximum, respectively. Ventilatory (diaphragm, intercostal, and transverse abdominis) and limb (gastrocnemius) muscles were excised at the end of the experimental period and examined for NOS activity and NOS protein expression. Neither submaximal nor maximum tracheal pressures were altered after 3 h of resistive loading. Diaphragmatic and intercostal muscle NOS activities declined significantly in response to moderate and severe loading, whereas those of transverse abdominis and gastrocnemius muscles remained unchanged. On the other hand, resistive loading had no significant effect on ventilatory and limb muscle NOS isoform expression. We propose that a contraction-induced decline in muscle NOS activity represents a compensatory mechanism through which muscle contractility and mitochondrial function are protected from the inhibitory influence of NO.     

Nitroxides increase the detectable amount of nitric oxide released from endothelial cells

Nitroxides are known to exert superoxide dismutase-mimetic properties and to decrease O-2- and H2O2-mediated cytotoxicity. However, the effect of nitroxides on .NO homeostasis has not been studied yet. The present study investigates the effect of nitroxides on the detectable amount of .NO released by 3-morpholinosydnonimine (SIN-1) and cultured endothelial cells. Cultured bovine aortic and atrial endothelial cells stimulated with 10 microM A23187 released a stable flux of .NO, as detected by .NO chemiluminescence. Addition of 100 units/ml SOD or 10 microM of the nitroxides 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPOL), 3-carboxy-proxyl, and 3-ethoxycarbonyl-proxyl, increased the chemiluminescence signal. The effect of these nitroxides on the amount of .NO released from cell monolayers was dose-dependent, with the highest efficacy between 30 and 100 microM. EPR spin trapping in SIN-1 solutions revealed the formation of .OH adducts from spontaneous dismutation of O-2 and concomitant reaction with H2O2. Both SOD and TEMPOL increased the signal intensity of the .OH adduct by accelerating the dismutation of O-2. The results of this study demonstrate that the SOD-mimetic activity of nitroxides increases the amount of bioavailable .NO in vitro.     

Serotonin inhibits nitric oxide synthesis in rat vascular smooth muscle cells stimulated with interleukin-1

The elimination half-life of fluoride is significantly increased in patients with chronic renal failure. This led us to conduct a study of variations of its plasma levels in 35 patients receiving dialysis treatment. In this population, there is a gaussian distribution of the values before and after the hemodialysis session, with a significant decrease in the averages. Furthermore, there is a highly significant correlation between fluoride levels before and after the dialysis session (P < 0.00001), and also between the amount of time in hemodialysis (in months) and the average fluoride level before dialysis (r = 0.624; P = 0.008). The presence of a group of patients consuming fluoride waters such as Vichy St-Yorre Water was easily identified by their excessive fluoride levels (above 100 micrograms/l), which could have a tendency to increase the risks of this group.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Electrochemical nitric oxide and nitrogen dioxide analyzer for use with inhaled nitric oxide

The use of inhaled nitric oxide (NO) in research and clinical applications requires the monitoring of NO and its autooxidation product nitrogen dioxide (NO2) in inspired gas and in the ambient environment. We describe an inexpensive electrochemical NO and NO2 analyzer that uses a critical orifice constant-flow controller and a microprocessor crossover correction for the measurement of NO and NO2 in the concentration range relevant to the use of inhaled NO. The analyzer proved to have good accuracy and precision for NO and NO2 in the range of concentrations relevant to studies of inhaled NO. In this range, the performance was similar to that of a chemiluminescence analyzer, and the response characteristics were not affected by varying the O2 concentration of the mixtures analyzed.     

Treatment of septic shock in rats with nitric oxide synthase inhibitors and inhaled nitric oxide

OBJECTIVE: To evaluate the effect of treatment with a combination of nitric oxide synthase inhibitors and inhaled nitric oxide on systemic hypotension during sepsis. DESIGN: Prospective, randomized, controlled study on anesthetized animals. SETTING: A cardiopulmonary research laboratory. SUBJECTS: Forty-seven male adult Sprague-Dawley rats. INTERVENTIONS: Animals were anesthetized, mechanically ventilated with room air, and randomized into six groups: a) the control group (C, n=6) received normal saline infusion; b) the endotoxin-treated group received 100 mg/kg i.v. of Escherichia coli lipopolysaccharide (LPS, n=9); c) the third group received LPS, and 1 hr later the animals were treated with 100 mg/kg i.v. Nw-nitro-L-arginine (LNA, n=9); d) the fourth group received LPS, and after 1 hr, the animals were treated with 100 mg/kg i.v. aminoguanidine (AG, n=9); e) the fifth group received LPS and 1 hr later was treated with LNA plus 1 ppm inhaled nitric oxide (LNA+NO, n=7); f) the sixth group received LPS and 1 hr later was treated with aminoguanidine plus inhaled NO (AG+NO, n=7). Inhaled NO was administered continuously until the end of the experiment. MEASUREMENTS AND MAIN RESULTS: Systemic mean blood pressure (MAP) was monitored through a catheter in the carotid artery. Mean exhaled NO (ENO) was measured before LPS (T0) and every 30 mins thereafter for 5 hrs. Arterial blood gases and pH were measured every 30 mins for the first 2 hrs and then every hour. No attempt was made to regulate the animal body temperature. All the rats became equally hypothermic (28.9+/-1.2 degrees C [SEM]) at the end of the experiment. In the control group, blood pressure and pH remained stable for the duration of the experiment, however, ENO increased gradually from 1.3+/-0.7 to 17.6+/-3.1 ppb after 5 hrs (p< .05). In the LPS treated rats, MAP decreased in the first 30 mins and then remained stable for 5 hrs. The decrease in MAP was associated with a gradual increase in ENO, which was significant after 180 mins (58.9+/-16.6 ppb) and reached 95.3+/-27.5 ppb after 5 hrs (p< .05). LNA and AG prevented the increase in ENO after LPS to the level in the control group. AG caused a partial reversal of systemic hypotension, which lasted for the duration of the experiment. LNA reversed systemic hypotension almost completely but only transiently for 1 hr, and caused severe metabolic acidosis in all animals. The co-administration of NO with AG had no added benefits on MAP and pH. In contrast, NO inhalation increased the duration of the reversal in MAP after LNA, alleviated the degree of acidosis, and decreased the mortality rate (from 55% to 29%). CONCLUSIONS: In this animal model, LPS-induced hypotension was alleviated slightly and durably after AG, but only transiently after LNA. Furthermore, co-administration of NO with AG had no added benefits but alleviated the severity of metabolic acidosis and mortality after LNA. We conclude that nitric oxide synthase (NOS) inhibitors, given as a single large bolus in the early phase of sepsis, can exhibit some beneficial effects. Administration of inhaled NO with NOS inhibitors provided more benefits in some conditions and therefore may be a useful therapeutic combination in sepsis. NO production in sepsis does not seem to be a primary cause of systemic hypotension. Other factors are likely to have a major role.     

Vascular smooth muscle nitric oxide synthase anomalies in Dahl/Rapp salt-sensitive rats

Salt-sensitive hypertension in the Dahl/Rapp rat (S strain) is prevented by L-arginine. Based on the observations that dexamethasone prevented the antihypertensive effect of L-arginine in these animals and the suggestion that a locus in or near an inducible nitric oxide synthase (NOS) gene on chromosome 10 cosegregated with hypertension in some F2 crosses that utilized the S rat, the present study explored the hypothesis that the vascular smooth muscle isoform of inducible NOS (NOS2) was abnormal in S rats. Primary cultures of aortic smooth muscle cells from S rats demonstrated impaired inducible NO production, which improved with increased L-arginine in the medium. Sequence analysis identified a single T-->C transversion that produced an amino acid substitution (S714P) between the FAD and FMN binding sites and a restriction fragment length polymorphism. This restriction fragment length polymorphism was present only in S rats. The mutation of NOS2 and the role of this enzyme in the pathogenesis of salt-sensitive hypertension in the Dahl/Rapp rat require further investigation.     

Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Nitric oxide (NO) is a pluripotent molecule that can be secreted by skeletal muscle through the activity of the neuronal constitutive isoform of NO synthase. To determine whether skeletal muscle and diaphragm might also express the macrophage-inducible form of NO synthase (iNOS) during provocative states, we examined tissue from mice at serial times after intravenous administration of Escherichia coli endotoxin. In these studies, iNOS mRNA was strongly expressed in the diaphragm and skeletal muscle of mice 4 h after intravenous endotoxin and was significantly diminished by 8 h after challenge. Induction of iNOS mRNA was followed by expression of iNOS immunoreactive protein on Western immunoblots. Increased iNOS activity was demonstrated by conversion of arginine to citrulline. Immunochemical analysis of diaphragmatic explants exposed to endotoxin in vitro revealed specific iNOS staining in myocytes, in addition to macrophages and endothelium. These results may be important in understanding the pathogenesis of respiratory pump failure during septic shock, as well as skeletal muscle injury during inflammation or metabolic stress.     

Is smooth muscle growth in primate arteries regulated by endothelial nitric oxide synthase?

PURPOSE: We investigated whether control of constitutive endothelial cell nitric oxide synthase (cNOS) and nitric oxide (NO) by changes in shear stress might be important for the regulation of smooth muscle cell (SMC) growth and vascular diameter. METHODS: Bilateral femoral arteriovenous fistulas were placed in baboons to increase the blood flow in the external iliac arteries. At 2 months, the fistula was ligated on one side to restore normal flow (flow switch). RESULTS: In response to flow switch and a decrease in shear stress, iliac artery lumenal area decreased and SMC proliferation was induced. A decline in NO production, cNOS messenger RNA (mRNA), and protein were associated with these biological effects. In a subset of animals with iliac arteries under high flow, infusion of N(omega)-nitro-L-arginine, an inhibitor of cNOS, did not induce proliferation. CONCLUSION: Shear stress can regulate cNOS, vasoconstriction, and SMC proliferation. A decrease in nitric oxide may be necessary, but is not sufficient to induce SMC proliferation in response to a decrease in blood flow.     

Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.     

Painful muscle cramps in liver cirrhosis and effects of oral taurine administration

We administered 3 g of taurine orally for four weeks to 35 patients suffering from liver cirrhosis with repeated muscle cramp (MC). Improvement of MC was noted in 22 cases (62.9%). We also determined the plasma taurine concentration in eight cases of liver cirrhosis with MC. The plasma taurine concentration before ingestion was 54.1 +/- 20.7 nmol/ml, whereas that of four weeks after ingestion was 125.1 +/- 59.1 nmol/ml, which was significantly elevated by 2.3 fold. As the concentration increased, the frequency of MC decreased, suggesting the good correlation between ingestion and the decrease in frequency of MC. In liver cirrhosis without MC the plasma taurine concentration was 81.0 +/- 16.7 nmol/ml, which was significantly higher than in liver cirrhosis with MC. In a few cases with taurine ingestion, serial plasma taurine concentrations were detected. Plasma taurine reached the peak value during the first week of ingestion and plasma taurine levels were maintained 2-5 fold higher during ingestion.