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     

Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide

Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.     

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS^–/–) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS^–/– cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N^G-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS^–/– cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS^–/– hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development. proliferation; heart development     

Mechanical stretch induces activation of skeletal muscle satellite cells in vitro.

Cultured quiescent satellite cells were subjected to mechanical stretch in a FlexerCell System. In response to stretch, satellite cells entered the cell cycle earlier than if they were under control conditions. Only a brief period of stretch, as short as 2 h, was necessary to stimulate activation. Additionally, conditioned medium from stretched cells could activate unstretched satellite cells. The presence of HGF on c-met-positive myogenic cells was detected by immunofluorescence at 12 h in culture, and immunoblots demonstrated that HGF was released by stretched satellite cells into medium. Also, stretch activation could be abolished by the addition of anti-HGF antibodies to stretched cultures, and activity in conditioned medium from stretched cells could be neutralized by anti-HGF antibodies. In addition, stretch appeared to cause release of preexisting HGF from the extracellular matrix. These experiments suggest that HGF may be involved in linking mechanical perturbation of muscle to satellite cell activation.     

Low-pH preparation of skeletal muscle satellite cells can be used to study activation in vitro

When skeletal muscle is stretched or injured, satellite cells are activated to enter the cell cycle, and this process could be mediated by hepatocyte growth factor (HGF) and nitric oxide (NO) as revealed by primary culture technique. In this system, which was originally developed by Allen et al. [Allen, R. E., Temm-Grove, C. J., Sheehan, S. M., & Rice, G. (1997). Skeletal muscle satellite cell cultures. Methods Cell Biol., 52, 155-176], however, some populations of satellite cells would receive activation signals during the cell isolation procedure; the high baseline level of activation diminishes the magnitude of the observed effect of HGF and NO. In this study, we modified the cell isolation procedure by lowering pH of muscle and isolation media from 7.2 (original) to 6.5. This modification was designed to block the activation signal generation, based on our previous observations that the satellite cell activation in response to mechanical stimulation only occurred between pH 7.1 and 7.5. Satellite cells prepared at low-pH showed a low baseline level of activation in bromodeoxyuridine incorporation and MyoD expression assays on control cultures, and demonstrated a large activation response to mechanical stretch, exogenous HGF and NO donor. Cell yield and myogenic purity were not affected by the modifications. The low-pH procedure could provide an improved satellite cell model for in vitro activation experiments.     

Activation of muscle satellite cells in single-fiber cultures.

Satellite stem cell activation is the process by which quiescent precursor cells resident on muscle fibers are recruited to cycle and move. Two processes are reported to affect satellite cell activation. In vivo, nitric oxide (NO) produced by NO synthase in fibers (NOS-Imu) promotes activation. In cell cultures, hepatocyte growth factor (HGF) is the major activating factor isolated from crushed muscle extract (CME). In this study we hypothesized that distinct and possibly related events were mediated by NO and HGF during activation. Intact fibers were cultured in the presence of bromodeoxyuridine (BrdU) to label DNA synthesis over 48 h. Experiments were designed to test the effects of CME, HGF, a NOS substrate L-arginine, and the NOS inhibitor L-NAME on activation, determined as the number of BrdU-positive satellite cells per fiber. Activation was increased significantly by CME, HGF, and L-arginine. L-Arginine increased activation in a dose-response manner. CME-induced activation was reduced significantly by NOS inhibition. Exposure to marcaine (10 min) caused reversible membrane damage without hypercontraction, as shown by characterizing the sarcolemmal integrity. The resulting decrease in satellite cell activation could be overcome by exogenous HGF. Results support the hypothesis that NO is involved in recruiting to cycle those satellite cells resident on fibers. Separate assessments of resident and free muscle cells showed that HGF and NO also participate in mobilizing satellite cells. Since HGF counteracted NOS inhibition and marcaine-induced membrane damage, data suggest that NO may mediate early steps in activation and precede HGF-mediated events.     

Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch

When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 μM (GsMTx-4), 10 μM (nifedipine), or 100 μM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 μM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade.     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Matrix metalloproteinase-2 mediates stretch-induced activation of skeletal muscle satellite cells in a nitric oxide-dependent manner

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, mediate HGF release from the matrix and this step in the pathway is downstream from NO synthesis [Yamada, M., Tatsumi, R., Kikuiri, T., Okamoto, S., Nonoshita, S., Mizunoya, W., et al. (2006). Matrix metalloproteinases are involved in mechanical stretch-induced activation of skeletal muscle satellite cells. Muscle Nerve, 34, 313-319]. Experiments reported herein provide evidence that MMP2 may be involved in the NO-dependent release of HGF in vitro. Whole lysate analyses of satellite cells demonstrated the presence of MMP2 mRNA and the protein. When rat satellite cells were treated with 30 microM sodium nitroprusside a NO donor or mechanical cyclic stretch for 2h period, inactive proMMP2 (72 kDa) was converted into 52-kDa form and this processing was abolished by adding a NO synthase inhibitor l-NAME (10 microM) to the stretch culture. The 52-kDa species was also generated by treatment of the recombinant MMP2 protein with 1 microM NOC-7 that can spontaneously release NO under physiological conditions without any cofactor, and its activating activity was demonstrated by applying the NOC-7-treated MMP2 to satellite cell culture. HGF release was detected in NOC-7-MMP2-conditioned media by western blotting; very little HGF was found in media that were generated from cultures receiving NOC-7-treated MMP2 (10 ng/ml) plus 250 ng/ml tissue inhibitor-1 of metalloproteinases. Therefore, results from these experiments provide evidence that NO-activated MMP2 may cause release of HGF from the extracellular matrix of satellite cells and contribute to satellite cell activation.     

Satellite cells are increasingly refractory to activation by nitric oxide and stretch in aged mouse-muscle cultures

Age-related muscle atrophy or sarcopenia results in progressive loss of muscle function and satellite cells in aging muscle are increasingly refractory to activation that could mitigate atrophy. We know that nitric oxide release triggered by mechanical stretch of skeletal muscle, initiates satellite cell activation in vitro in single fiber, single cell and whole-muscle cultures, and in vivo in animals. This study examined muscle cell activation using tritiated-thymidine incorporation into the DNA of muscle cells in cultured muscles from female mice between 6 weeks and 18 months-of-age. Experiments examined age-related changes in activation by mechanical stretch and/or NO treatments (with the substrate of nitric oxide synthase (l-arginine), a nitric oxide donor (isosorbide dinitrate) and/or nitric oxide synthase inhibition). Activation without stretch was highest at 8 months. Stretching muscles by 10% more than doubled activation in muscles at 6 weeks of age and only a 20% stretch similarly activated cells in cultured 6-month-old muscles. Only treatment with ISDN in combination with a 20% stretch activated cell proliferation in muscles from 8-month-old mice. A nitric-oxide donor drug rescued muscle satellite cells in adult, 8-month-old mice from being refractory to mechanical stretch, apparently by overcoming an ineffective release of nitric oxide during stretch. Results suggest that treatment with nitric oxide has the potential to enhance the effectiveness of exercise in preventing the onset of age-related muscle atrophy in adult muscle.     

Role of NO and PAF in the impairment of skeletal muscle contractility induced by TNF-alpha

The role of platelet-activating factor (PAF) and nitric oxide (NO) as mediators of the effects of tumor necrosis factor-alpha (TNF-alpha) on skeletal muscle contractility was studied in guinea pig extensor digitorum longus (EDL) muscle. TNF-alpha (5-10 ng/ml) reduced contractility at every stimulation frequency (1-200 Hz) and shifted the force-frequency relationship to the right. The role of NO and PAF as mediators of TNF-alpha was suggested by the protective effect of N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mM), but not of N(G)-nitro-D-arginine methyl ester (D-NAME; 1 mM), and by the inhibitory effect of the PAF-receptor antagonist WEB-2170 (3 microM). TNF-alpha increased the production of PAF and NO. Similar to TNF-alpha, both S-nitroso-N-acetylpenicillamine (0.5-1 microM), an NO-generating compound, and PAF (10-20 nM) reduced EDL contractility. L-NAME, but not D-NAME, blocked the negative effect of PAF. Blockade of phospholipase A(2), which is required for PAF synthesis, significantly reduced the effects of TNF-alpha. WEB-2170 inhibited NO synthesis induced by TNF-alpha and PAF-stimulated NO production. These results suggest that both PAF and NO contribute to the development of the mechanical alterations induced by TNF-alpha and that NO production is downstream to the synthesis of PAF.     

Cyclosporine A-increased nitric oxide production in the rat dorsal hippocampus mediates convulsions

To test whether nitric oxide (NO) participates in cyclosporine A (CsA)-induced neurotoxicity including convulsions, we examined the effect of an NO synthase inhibitor on convulsions induced by combined treatment with CsA and bicuculline in mice and the effect of CsA on NO production in the dorsal hippocampus using an in vivo microdialysis method in rats. CsA (200 mg/kg, i.p.) significantly increased the intensity of convulsions induced by an intracerebroventricular injection of bicuculline (25 pmol) in mice. This facilitation was blocked by N omega -nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, but not by N omega -nitro-D-arginine methyl ester (D-NAME), an inactive form of L-NAME (10 mg/kg, i.p.). CsA (20-50 mg/kg, i.p.) dose-dependently increased NO 2 - levels in dialysates obtained with microdialysis in the rat dorsal hippocampus. This enhanced NO 2 - formation was blocked by L-NAME but not by D-NAME (50 mg/kg, i.p.). These findings suggest that CsA stimulates NO production and induces convulsions as a result of an interaction between NO and the gamma-aminobutyric acid (GABA) system in the hippocampus.     

Long-term administration of D-NAME induces hemodynamic and structural changes in the cardiovascular system

N(G)-nitro-D-arginine-methyl ester (D-NAME) is considered to be an inactive enantiomer of L-NAME and is generally used as the negative control for NO synthase inhibition with L-NAME. With the aim to compare the effects of 4-week L-NAME and D-NAME treatments on hemodynamic and cardiovascular structural parameters, four groups of male Wistar rats were investigated: the controls and groups administered 40 and 20 mg/kg/day of L-NAME and 40 mg/kg/day of D-NAME. At the end of the experiment, myocardial NO synthase activity decreased by 42, 24 and 25%; aortic NO synthase activity decreased by 35, 15 and 13% vs. controls in the L-NAME 40, L-NAME 20 and D-NAME 40 groups, respectively. The DNA concentrations in the myocardium and the aorta increased significantly after L-NAME and D-NAME treatments. The inhibition of NO synthase was accompanied by a significant elevation in systolic blood pressure in all three groups. The LVW/BW ratio increased by 27, 14 and 13% vs. controls in the L-NAME 40, L-NAME 20 and D-NAME 40 groups, respectively. The aortic wall mass, measured as the cross-sectional area, increased by 45, 17 and 25% vs. controls in the L-NAME 40, L-NAME 20 and D-NAME 40 groups, respectively. Myocardial fibrosis represented 0.94% in the controls, but 7.96, 4.70 and 5.25% in L-NAME 40, L-NAME 20 and D-NAME 40 groups, respectively. It is concluded that D-NAME, although less affective than L-NAME, inhibits NO synthase activity resulting in hemodynamic and structural changes in the cardiovascular system similar to the changes induced by half the dose of L-NAME. Thus, the consideration of D-NAME as an inactive enantiomer and its use as the negative control needs to be reevaluated.     

Structural alterations in the heart after long-term L-NAME and D-NAME treatment

N(G)-nitro-D-arginine methyl ester (D-NAME), considered as an inactive enantiomer of NAME, is generally used as a negative control for NO synthase inhibition with L-NAME. The aim of this work was to compare the effect of L-NAME (20 and 40 mg/kg/day), and D-NAME (40 mg/kg/day) on hemodynamic and structural parameters in the rat cardiovascular system. After 4 weeks of treatment, blood pressure and left ventricle weight/body weight ratio increased significantly in all studied groups versus control. Myocardial fibrosis (in %) represented 0.94 +/- 0.04 in control, 4.70 +/- 0.39 in L-NAME (20 mg/kg/day), 10.54 +/- 0.91 in L-NAME (40 mg/kg/day) and 5.25 +/- 0.46 in D-NAME (40 mg/kg/day) group. We conclude that in a long-term experiment D-NAME provokes similar changes in cardiovascular system like L-NAME.     

Further evidence for the role of nitric oxide in maternal aggression: effects of L-NAME on maternal aggression towards female intruders in Wistar rats

It has been shown that nitric oxide (NO) increases aggression in male mice, whereas it decreases aggression in lactating female mice and prairie voles. It is also known that aggression can be exhibited at different levels in rodent species, strain or subtypes. The aims of this study were to investigate the proportion of aggressiveness in Wistar rats, the effect of intraperitoneally administered nonspecific nitric oxide synthase (NOS) inhibitor L-NAME (NG-nitro L-arginine methyl ester) on maternal aggression towards female intruders, and whether these effects are due to NO production or not. Rats were given saline intraperitoneally on the postpartum Day 2 and aggression levels were recorded. The same rats were given 60 mg/kg L-NAME or D-NAME (NG-nitro D-arginine methyl ester) on the postpartum Day 3 and their effects on aggression levels were compared to saline. While L-NAME administration did not cause any differences in the total number of aggressive behavior, aggression duration and aggression intensity, it reduced the proportion of animals showing aggressive behavior. In addition, the latency of the first aggression was significantly increased by L-NAME. In the D-NAME group, however, no significant change was found. Our results have shown that L-NAME reduces maternal aggression towards female intruders in Wistar rats through inhibition of NO production. These results suggest that the role of NO in offensive and defensive maternal aggression shares neural mechanisms.     

Peripheral or central administration of nitric oxide synthase inhibitor affects feeding behavior in chicks

We investigated the effect of peripheral or central administration of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on food intake in layer and broiler chicks (Gallus gallus). The intraperitoneal administration of L-NAME significantly decreased food intake in both broiler and layer chicks while the administration of D-NAME, an inactive form of L-NAME, had no effect. The intracerebroventricular (ICV) injection of L-NAME did not affect food intake in broiler chicks. However, ICV injection of L-NAME increased food intake in layer chicks while the injection of D-NAME had no effect. In addition to this, L-NAME-induced feeding was negated with the co-injection of L-arginine, suggesting that NO acts as a feeding-inhibitor signal in the brain of layer chicks. The present study revealed that administration of NO synthase inhibitor affected food intake in chicks, but the effect might be changed by chick strain and position of the injection.     

Expression of nitric oxide synthase isoforms in normal ventilatory and limb muscles

Hussain, Sabah N. A., Qasim El-Dwairi, Mohammed N. Abdul-Hussain, and Dalia Sakkal. Expression of nitric oxidesynthase isoforms in normal ventilatory and limb muscles.J. Appl. Physiol. 83(2): 348-353, 1997.Nitric oxide (NO), an important messenger molecule withwidespread actions, is synthesized by NO synthases (NOS). In thisstudy, we investigated the correlation between fiber type and NOSactivity among ventilatory and limb muscles of various species. We alsoassessed the presence of the three NOS isoforms in normal skeletalmuscles and how various NOS inhibitors influence muscle NOS activity.NOS activity was detected in various muscles; however, NOS activity inrabbits and rats varied significantly among different muscles.Immunoblotting of muscle samples indicated the presence of both theneuronal NOS and the endothelial NOS isoforms but not thecytokine-inducible NOS isoform. However, these isoforms were expressedto different degrees in various muscles. Although the neuronal NOSisoform was detectable in the canine diaphragm, very weak expressionwas detected in rabbit, rat, and mouse diaphragms. The endothelial NOSisoform was detected in the rat and mouse diaphragms but not in thecanine and rabbit diaphragms. We also found thatN^G-nitro-L-arginine methyl ester,7-nitroindazole, andS-methylisothiourea werestronger inhibitors of muscle NOS activity than was aminoguanidine. These results indicate the presence of different degrees ofconstitutive NOS expression in normal ventilatory and limb muscles ofvarious species. Our data also indicate that muscle NOS activity is not determined by fiber type distribution but by other not yet identified factors. The functional significance of this expression remains to beassessed.

     

Nitric oxide decreases lung injury after intestinal ischemia

Terada, Lance S., Nancy N. Mahr, and Eugene D. Jacobson.Nitric oxide decreases lung injury after intestinal ischemia. J. Appl. Physiol. 81(6):2456-2460, 1996.After injury to a primary organ, mediators arereleased into the circulation and may initiate inflammation of remoteorgans. We hypothesized that the local production of nitric oxide (NO)may act to limit the spread of inflammation to secondarily targetedorgans. In anesthetized rats, 30 min of intestinal ischemia followed by2 h of reperfusion (I/R) did not increase lung albumin leak. However,after treatment with N^G-nitro-L-arginine methyl ester(L-NAME), intestinal I/R led to increased lung leak, suggesting a protective effect of endogenous NO.The site of action of NO appeared to be the lung and not the gutbecause 1) after treatment withL-NAME, local delivery of NO tothe lung by inhalation abolished the increase in intestinal I/R-inducedlung leak; 2)L-NAME had no effect onepithelial permeability (⁵¹Cr-labeled EDTA clearance) ofreperfused small bowel; and 3) after treatment with L-NAME, localdelivery of NO to the gut by luminal perfusion did not improveepithelial permeability of reperfused intestines. Furthermore,L-NAME increased, and inhaled NOde- creased, the density of lung neutrophils in rats subjected to intestinal I/R, and treatment with the selectin antagonist fucoidan abolished L-NAME-induced lungleak in rats subjected to intestinal I/R. We conclude thatendogenous lung NO limits secondary lung injury after intestinal I/R bydecreasing pulmonary neutrophil retention.

     

The contribution of muscle progenitor cells to maintaining morphological characteristics of unweighted rat soleus muscle during passive stretch

Skeletal muscle work hypertrophy is usually connected with muscle progenitor SC (satellite cells) activation with subsequent incorporation their nuclei into myofibers. Passive stretch of unloaded muscle was earlier established to prevent atrophic processes and be accompanied by enhanced protein synthesis. We hypothesized that elimination of SC proliferation capacity by gamma-irradiation would partly preavent stretched muscle fiber capability to maintain their size under condition of gravitational unloading. To assess the role of muscle progenitor (satellite) cells in development of passive stretch preventive effect SC proliferation was suppressed by local exposure to ionizing radiation (2500 Rad) and then subsequent hindlimb suspension or hindlimb suspension with concomitant passive stretch were carried out. Reduction of myofiber cross-sectional area and decrease in myo-nuclei number accompanying unloaded muscle atrophy were completely abolished by passive stretch both in irradiated and sham-treated animals. We concluded that satellite cells did not make essential contribution to passive stretch preventive action under condition of simulated weightlessness.