Estimating oxygen consumption rates of arteriolar walls under physiological conditions in rat skeletal muscle

To examine the effects of vascular tone reduction on O2 consumption of the vascular wall, we determined the O2 consumption rates of arteriolar walls under normal conditions and during vasodilation induced by topical application of papaverine. A phosphorescence quenching technique was used to quantify intra- and perivascular PO2 in rat cremaster arterioles with different branching orders. Then, the measured radial PO2 gradients and a theoretical model were used to estimate the O2 consumption rates of the arteriolar walls. The vascular O2 consumption rates of functional arterioles were >100 times greater than those observed in in vitro experiments. The vascular O2 consumption rate was highest in first-order (1A) arterioles, which are located upstream, and sequentially decreased downstream in 2A and 3A arterioles under normal conditions. During papaverine-induced vasodilation, on the other hand, the O2 consumption rates of the vascular walls decreased to similar levels, suggesting that the high O2 consumption rates of 1A arterioles under normal conditions depend in part on the workload of the vascular smooth muscle. These results strongly support the hypothesis that arteriolar walls consume a significant amount of O2 compared with the surrounding tissue. Furthermore, the reduction of vascular tone of arteriolar walls may facilitate an efficient supply of O2 to the surrounding tissue.     

Nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle ischemia-reperfusion injury.

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia-reperfusion (IR) injury to rat skeletal muscle in vivo. Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitor N-nitro-L-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitor S-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosis in vitro did not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.     

Nitric oxide and repair of skeletal muscle injury

The muscle wound healing occurs in three overlapping phases: (1) degeneration and inflammation, (2) muscle regeneration, and (3) fibrosis. Simultaneously to injury cellular infiltration by neutrophils and macrophages occur, as well as cellular ‘respiratory burst’ via activation of the enzyme NADPH oxidase. When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle, divide, differentiate and fuse with muscle fibers to repair damaged regions and to enhance hypertrophy of muscle fibers. This process depends on nitric oxide (NO) production, metalloproteinase (MMP) activation and release of hepatocyte growth factor (HGF) from the extracellular matrix. Generation of a fibrotic scar tissue, with partial loss of function, can also occur, and seems to be dependent, at least in part, on local TGF-β expression, which can be downregulated by NO. Hence, regeneration the muscle depends on the type and severity of the injury, the appropriate inflammatory response and on the balance of the processes of remodeling and fibrosis. It appears that in all these phases NO exerts a significant role. Better comprehension of this role, as well as of the participation of other important mediators, may lead to development of new treatment strategies trying to tip the balance in favor of greater regeneration over fibrosis, resulting in better functional recovery.     

AICAR inhibits oxygen consumption by intact skeletal muscle cells in culture

Activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) with aminoimidazole carboxamide ribonucleotide (AICAR) increases skeletal muscle glucose uptake and fatty acid oxidation. The purpose of these experiments was to utilize AICAR to enhance palmitate consumption by mitochondria in cultured skeletal muscle cells. In these experiments, we treated C2C12 myotubes or adult single skeletal muscle fibers with varying concentrations of AICAR for different lengths of time. Surprisingly, acute AICAR exposure at most concentrations (0.25–1.5 mM), but not all (0.1 mM), modestly inhibited oxygen consumption even though AICAR increased AMPK phosphorylation. The data suggest that AICAR inhibited oxygen consumption by the cultured muscle in a non-specific manner. The results of these experiments are expected to provide valuable information to investigators interested in using AICAR in cell culture studies.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide caninhibit cytoskeletal breakdown in skeletal muscle cells by inhibitingcalpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore onC₂C₁₂ muscle cells, including preventing talinproteolysis and release into the cytosol and reducing loss of vinculin,cell detachment, and loss of cellular protein. These results indicatethat nitric oxide inhibition of calpain protected the cells fromionophore-induced proteolysis. Calpain inhibitor I and a cell-permeablecalpastatin peptide also protected the cells from proteolysis,confirming that ionophore-induced proteolysis was primarily calpainmediated. The activity of m-calpain in a casein zymogram was inhibitedby sodium nitroprusside, and this inhibition was reversed bydithiothreitol. Previous incubation with the active site-targetedcalpain inhibitor I prevented most of the sodium nitroprusside-inducedinhibition of m-calpain activity. These data suggest that nitric oxideinhibited m-calpain activity via S-nitrosylation of the active sitecysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

     

Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells

In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C₂C₁₂ myotubes were subjected to a single 10% step stretch for 0–4 days. The apparent elastic modulus of the cells, E_app, was subsequently determined by atomic force microscopy. Static stretch led to significant increases (P < 0.01) in E_app beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor L-arginine onto stretched cells further enhanced E_app, NOS activity, and nNOS expression, whereas the presence of the NO inhibitor N-nitro-L-arginine methyl ester (L-NAME) reversed the effects of mechanical stimulation and of L-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with L-NAME showed a significant decrease in E_app, whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in E_app. Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis. mechanical stimulation; apparent elastic modulus; skeletal muscle cells; nitric oxide; stretch     

Local and remote arteriolar dilations initiated by skeletal muscle contraction

To investigate the relationship between skeletal muscle metabolism and arteriolar dilations in the region local to contracting muscle fibers as well as dilations at remote arteriolar regions upstream, we used a microelectrode on cremaster muscle of anesthetized hamsters to stimulate four to five muscle fibers lying approximately perpendicular to and overlapping a transverse arteriole. Before, during, and after muscle contraction, we measured the diameter of the arteriole at the site of muscle fiber overlap (local) and at a remote site approximately 1,000 microm upstream. Two minutes of 2-, 4-, or 8-Hz stimulation (5-10 V, 0.4-ms duration) produced a significant dilation locally (8.2 +/- 2.0-, 22.5 +/- 2.4-, and 30.9 +/- 2.1-microm increase, respectively) and at the remote site (4.2 +/- 0.8, 11.0 +/- 1.1, and 18.9 +/- 2.7 microm, respectively). Muscle contraction at 4 Hz initiated a remote dilation that was unaffected by 15-min micropipette application of either 2 microM tetrodotoxin, 0.07% halothane, or 40 microM 18-beta-glycyrrhetinic acid between the local and upstream site. Therefore, at the arteriolar level, muscle contraction initiates a robust remote dilation that does not appear to be transmitted via perivascular nerves or gap junctions.     

Nitric oxide mediated oxidative stress injury in rat skeletal muscle subjected to ischemia/reperfusion as evaluated by chemiluminescence.

The involvement of nitric oxide (*NO) in oxidative stress in the rat gastrocnemius muscle subjected to ischemia/reperfusion injury was investigated using a specific and sensitive chemiluminescence (CL) method for measurement of both membrane lipid peroxide and total tissue antioxidant capacity (TRAP). In addition, inhibitors of nitric oxide synthase enzymes were used. The CL time-course curve increased dramatically after 1, 2, and 4 h of reperfusion, reaching values about 12 times higher than those of both control and ischemic rats. Initial velocity (V0) increased from 13.6 cpm mg protein(-1) min(-1) in the ischemic group, to 7341-8524 cpm mg protein(-1) min(-1) following reperfusion. The administration of L-NAME prior to reperfusion significantly reduced (p<0.007) the time-course of the CL curve, decreasing the V(0) value by 51% and preventing antioxidant consumption for 1h following reperfusion. No significant change in CL time-course curve and TRAP values were observed with aminoguanidine treatment. On contrary, after 4h following reperfusion, pre treatment with aminoguanidine led to a significant decrease (p < 0.0001) in the time-course of the CL curve, where V0 decreased by 75% and TRAP returned to control levels. No significant change in CL time-course curve and TRAP values were observed with L-NAME treatment. When RT-PCR was carried out with an iNOS-specific primer, a single band was detected in RNA extracted from muscle tissue of only the 4 h ischemia/4 h reperfusion group. No bands were found in either the control, 4 h ischemia or 4 h ischemia/1 h reperfusion groups. Based on these results, we conclude that *NO plays an important role in oxidative stress injury, possibly via -ONOO, in skeletal muscle subjected to ischemia/reperfusion. Our results also show that cNOS isoenzymes are preferentially involved in *NO generation at the beginning of reperfusion and that iNOS isoenzyme plays an important role in reperfusion injury producing *NO later in the process.     

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

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

Nitric oxide does not modulate whole body oxygen consumption in anesthetized dogs.

The effects of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and the NO donor sodium nitroprusside (SNP) on whole body O2 consumption (VO2) were assessed in 16 dogs anesthetized with fentanyl or isoflurane. Cardiac output (CO) and mean arterial pressure (MAP) were measured with standard methods and were used to calculate VO2 and systemic vascular resistance (SVR). Data were obtained in each dog under the following conditions: 1) Control 1, 2) SNP (30 microg. kg-1. min-1 iv) 3) Control 2, 4) L-NAME (10 mg/kg iv), and 5) SNP and adenosine (30 and 600 microg. kg-1. min-1 iv, respectively) after L-NAME. SNP reduced MAP by 29 +/- 3% and SVR by 47 +/- 3%, while it increased CO by 39 +/- 9%. L-NAME had opposite effects; it increased MAP and SVR by 24 +/- 4% and 103 +/- 11%, respectively, and it decreased CO by 37 +/- 3%. Neither agent changed VO2 from the baseline value of 4.3 +/- 0.2 ml. min-1. kg-1, since the changes in CO were offset by changes in the arteriovenous O2 difference. Both SNP and adenosine returned CO to pre-L-NAME values, but VO2 was unaffected. We conclude that 1) basally released endogenous NO had a tonic systemic vasodilator effect, but it had no influence on VO2; 2) SNP did not alter VO2 before or after inhibition of endogenous NO production; 3) the inability of L-NAME to increase VO2 was not because CO, i.e., O2 supply, was reduced below the critical level.     

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

Nitric oxide modulates sensitivity to ABA

Nitric oxide (NO) is a gas with crucial signaling functions in plant defense and development. As demonstrated by generating a triple nia1nia2noa1-2 mutant with extremely low levels of NO (February 2010 issue of Plant Physiology), NO is synthesized in plants through mainly two different pathways involving nitrate reductase (NR/NIA) and NO Associated 1 (AtNOA1) proteins. Depletion of basal NO levels leads to a priming of ABA-triggered responses that causes hypersensitivity to this hormone and results in enhanced seed dormancy and decreased seed germination and seedling establishment in the triple mutant. NO produced under non-stressed conditions represses inhibition of seed developmental transitions by ABA. Moreover, NO plays a positive role in post-germinative vegetative development and also exerts a critical control of ABA-related functions on stomata closure. The triple nia1nia2noa1-2 mutant is hypersensitive to ABA in stomatal closure thus resulting in a extreme phenotype of resistance to drought. In the light of the recent discovery of PYR/PYL/RCAR as a family of potential ABA receptors, regulation of ABA sensitivity by NO may be exerted either directly on ABA receptors or on downstream signalling components; both two aspects that deserve our present and future attention.Key words: nitric oxide, abscisic acid, seed germination, stomata openingPlant development is the result of the succesfull execution of several programs that control the transition between different growth phases. Every developmental transition is regulated through coordinated mechanisms that involved exogenous environmental factors such as light and temperature as well as endogenous cues, including levels of primary and secondary metabolites. Among the latter, hormones such as gibberellins (GA), auxins, citokinins, ethylene and abscisic acid (ABA) participate in the control of most of the developmental transitions.^1,2 During the last years, nitric oxide (NO) has gained an increasing role as an essential player in plant defense responses³ as well as a co-regulator of many developmental processes.⁴ However, studies of NO function as a regulatory molecule in plants have been hampered by the scanty, limited and controversial knowledge on how this gas is synthesized in plants.^5,6 This situation has moved researchers in this area to adopt pharmacological approaches based on chemicals acting as artificial NO donors as well as inhibitors or scavengers of NO action. The lack of specificity and the inherent artificial effects of these chemicals can be overcome by genetic approaches based on the use of mutants with endogenous low levels of NO. In February 2010 issue of Plant Physiology, we report the generation and further characterization of a triple nia1nia2noa1-2 mutant that contains extremely low levels of NO due to the impairment of two NO biosynthetic pathways involving nitrate reductase (NIA/NR) or NO Associated 1 (AtNOA1) proteins.⁷ These findings support that NO is mainly produced through those pathways in Arabidopsis. However, the possible existence of a minor still uncharacterized pathways involved in the residual production of NO can not be ruled out at this time.Further functional characterization of nia1nia2noa1-2 mutant in terms of development has pointed to NO as an overall positive regulator of plant growth, affecting to almost every developmental stage from seed germination to reproductive development. Accordingly, triple mutant plants display a delayed growth resulting in small shoot and root size and they also produce low amounts of viable seeds.⁷Dormancy and seed germination are developmental programs largely regulated by the combined action of GA and ABA.¹ GA promote breaking of dormancy and promote germination whereas ABA acts as a brake in those processes, thus ensuring a timely seed germination. Our data from the characterization of dormancy and seed germination in the nia1nia2noa1-2 mutant suggest that NO’s role in the control of those processes may be exerted through modulation of the sensitivity to ABA (Fig. 1A). Seeds from NO deficient plants have increased dormancy and lower seed germination and seedling establishment rates than wild type seeds due to the enhanced ABA inhibitory action. These effects can be reversed by exogenous application of NO to nia1nia2noa1-2, suggesting that the sensitivity to ABA is actually controlled by the endogenous levels of NO. The recent identification of PYR/PYL/RCAR family of ABA receptors,^8,9 and the further characterization of the essential ABA regulatory module including receptor, protein phosphatases of the 2C class and kinases of the SnRK2 family¹⁰ point to these components as potential targets of NO in regulating sensitivity to ABA (Fig. 1B). This work is in progress in our lab but we already know that some of the PYR/PYL/RCAR receptors and SnRKs are actually regulated by NO and also that this regulation may be exerted at different levels (Lozano-Juste J and León J, unpublished data).Open in a separate windowFigure 1Interactions between NO and ABA results in modulated sensitivity to ABA throughout development. (A) NO synthesized through nitrate reductase (NR/NIA) and NO associatedI (AtNOA1) protein regulate germinative and post-germinative development as well as stomata movements through modulation of the sensitivity to ABA. Arrows and bars represent positive and negative effects, and the thickness of lines are proportional to the magnitud of regulatory effects. (B) Scheme of a minimal ABA signalling module and the potential targets of NO. Dashed lines represent effects still to be demonstrated. (C) ABA signalling in stomata guard cells through Ca²⁺-dependent and -independent pathways and the potential interactions with NO as represented by dashed lines.The enhanced sensitivity to ABA observed in germinative and post-germinative development of nia1nia2noa1-2, is extended throughout plant life cycle and it is actually the cause of the very strong resistance of nia1nia2noa1-2 plants to water deficit conditions.⁷ Stomatal aperture is a fine-tuned process controlled mainly through a balance between the light-promoted opening and the ABA-mediated promotion of closure and inhibition of opening¹¹ (Fig. 1A). It has been previously reported that ABA function on stomata movements involve the participation of NO as well as Ca²⁺ in such a way that Ca²⁺ chelators and NO scavengers block ABA action on stomata movements.¹² Stomata of nia1nia2noa1-2 leaves, despite of being depleted of NO, are not impaired for ABA inhibition of stomata opening but, in turn, they seem to be primed for a more efficient ABA response (Fig. 1A). Contrary to the Ca²⁺ requirements for ABA action on wild type stomata movements, this process is not affected by Ca²⁺ chelators in nia1nia2noa1-2 stomata, and it thus seems to be independent of Ca²⁺ in NO-deficient backgrounds (Fig. 1C). As mentioned above, NO might regulate sensitivity to ABA by acting on ABA receptors or on SnRKs, some of which are Ca²⁺-independent kinases. Both receptors and Ca^2+--independent kinases are likely targets of NO in the modulation of stomata sensitivity to ABA thus explaining the more efficient stomata closure in nia1nia2noa1-2 leaves, and the consequent low rates of evapotranspiration that leads to the extreme resistance of triple mutant plants to drought.The future characterization of the interactions between NO and key components of ABA signaling will be the basis for a better knowledge of the functional interactions between different hormones in plant development and defense, but it will also open up new possibilities of identifying new targets and strategies leading to improved drought resistance.     

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.