Evidence that nitric oxide increases glucose transport in skeletal muscle

Balon, Thomas W., and Jerry L. Nadler. Evidence thatnitric oxide increases glucose transport in skeletal muscle.J. Appl. Physiol. 82(1): 359-363, 1997.Nitric oxide synthase (NOS) is expressed in skeletal muscle.However, the role of nitric oxide (NO) in glucose transport in thistissue remains unclear. To determine the role of NO in modulatingglucose transport, 2-deoxyglucose (2-DG) transport was measured in ratextensor digitorum longus (EDL) muscles that were exposed to either amaximally stimulating concentration of insulin or to an electricalstimulation protocol, in the presence ofN^G-monomethyl-L-arginine,a NOS inhibitor. In addition, EDL preparations were exposed to sodiumnitroprusside (SNP), an NO donor, in the presence of submaximal andmaximally stimulating concentrations of insulin. NOS inhibition reducedboth basal and exercise-enhanced 2-DG transport but had no effect oninsulin-stimulated 2-DG transport. Furthermore, SNP increased 2-DGtransport in a dose-responsive manner. The effects of SNP and insulinon 2-DG transport were additive when insulin was present inphysiological but not in pharmacological concentrations. Chronictreadmill training increased protein expression of both type I and typeIII NOS in soleus muscle homogenates. Our results suggest that NO maybe a potential mediator of exercise-induced glucose transport.

     

Endotoxin-induced skeletal muscle contractile dysfunction: contribution of nitric oxide synthases

The aims of thisstudy were to assess the role of nitric oxide (NO) and the contributionof different NO synthase (NOS) isoforms in skeletal muscle contractiledysfunction in septic shock. Four groups of conscious rats wereexamined. Group 1 served as control; groups 2, 3, and4 were injected withEscherichia coli endotoxin [lipopolysaccharide (LPS), 20 mg/kg ip] and killed after 6, 12, and 24 h, respectively. Protein expression was assessed byimmunoblotting and immunostaining. LPS injection elicited a transientexpression of the inducible NOS isoform, which peaked 12 h after LPSinjection and disappeared within 24 h. This expression coincided with a significant increase in nitrotyrosine formation (peroxynitrite footprint). Muscle expression of the endothelial and neuronal NOSisoforms, by comparison, rose significantly and remained higher thancontrol levels 24 h after LPS injection. In vitro measurement of musclecontractility 24 h after LPS injection showed that incubation with NOSinhibitor (S-methyliosothiourea)restored the decline in submaximal force generation, whereas maximalmuscle force remained unaffected. We conclude that NO plays asignificant role in muscle contractile dysfunction in septic animalsand that increased NO production is due to induction of the inducibleNOS isoform and upregulation of constitutive NOS isoforms.

     

MKP-1 switches arginine metabolism from nitric oxide synthase to arginase following endotoxin challenge

L-Arginine (L-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and L-ornithine (L-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing L-arg metabolism from NO production to L-orn production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and Mkp-1^–/– mice, as well as in vivo in wild-type and Mkp-1^–/– mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases. lung injury; macrophage; ornithine; mitogen-activated protein kinases     

Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes

The effects of nitric oxide (NO) produced by cardiac inducibleNO synthase (iNOS) on myocardial injury after oxidative stress wereexamined. Interleukin-1 induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS,L-arginine enhanced NOproduction in a concentration-dependent manner. Glutathione peroxidase(GPX) activity in myocytes was attenuated by elevated iNOS activity andby an NO donor,S-nitroso-N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition ofH₂O₂(0.1 mM, 1 h). Inhibition of iNOS withN-nitro-L-argininemethyl ester ameliorated the effects of NO-enhancing treatments onmyocardial injury and GPX activity. SNAP augmented the myocardialinjury induced byH₂O₂.Inhibition of GPX activity with antisense oligodeoxyribonucleotide forGPX mRNA increased myocardial injury byH₂O₂.Results suggest that the induction of cardiac iNOS promotes myocardialinjury due to oxidative stress via inactivation of the intrinsicantioxidant enzyme, GPX.

     

Regulation of nitric oxide production in response to skeletal muscle activation

Nitric oxide(NO) is synthesized in normal muscle fibers by the neuronal (nNOS) andthe endothelial (ecNOS) isoforms of nitric oxide synthase (NOS). NOcontributes to the regulation of several processes such asexcitation-contraction coupling and mitochondrial respiration. Weassessed in this study whether NO production is regulated in responseto an acute increase in muscle activation. Three groups ofanesthetized, tracheostomized, spontaneously breathing rats wereexamined after an experimental period of 3 h. Group 1 served as a control (no loading), whereasgroups 2 and3 were exposed to moderate and severeinspiratory resistive loads, respectively, which elicited trachealpressures of 30 and 70% of maximum, respectively. Ventilatory(diaphragm, intercostal, and transverse abdominis) and limb(gastrocnemius) muscles were excised at the end of the experimentalperiod 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 NOSactivities declined significantly in response to moderate and severeloading, whereas those of transverse abdominis and gastrocnemiusmuscles remained unchanged. On the other hand, resistive loading had nosignificant effect on ventilatory and limb muscle NOS isoformexpression. We propose that a contraction-induced decline in muscle NOSactivity represents a compensatory mechanism through which musclecontractility and mitochondrial function are protected from theinhibitory influence of NO.

     

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo

Reiser, Peter J., William O. Kline, and Pal L. Vaghy.Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo. J. Appl. Physiol. 82(4): 1250-1255, 1997.Fast-twitch skeletal muscles contain more neuronal-type nitricoxide synthase (nNOS) than slow-twitch muscles because nNOS is presentonly in fast (type II) muscle fibers. Chronic in vivo electricalstimulation of tibialis anterior and extensor digitorum longus musclesof rabbits was used as a method of inducing fast-to-slow fiber typetransformation. We have studied whether an increase in musclecontractile activity induced by electrical stimulation alters nNOSexpression, and if so, whether the nNOS expression decreases to thelevels present in slow muscles. Changes in the expression of myosinheavy chain isoforms and maximum velocity of shortening of skinnedfibers indicated characteristic fast-to-slow fiber type transformationafter 3 wk of stimulation. At the same time, activity of NOS doubled inthe stimulated muscles, and this correlated with an increase in theexpression of nNOS shown by immunoblot analysis. These data suggestthat nNOS expression in skeletal muscle is regulated by muscle activityand that this regulation does not necessarily follow the fast-twitchand slow-twitch pattern during the dynamic phase of phenotypetransformation.

     

Cytochrome c mRNA in skeletal muscles of immobilized limbs

Booth, Frank W., Wei Lou, Marc T. Hamilton, and Zhen Yan.Cytochrome c mRNA in skeletalmuscles of immobilized limbs. J. Appl.Physiol. 81(5): 1941-1945, 1996.Even thoughimmobilization of a slow skeletal muscle in a lengthened positionprevents muscle atrophy, it is unknown whether this treatment wouldprevent a decrease in mitochondrial quantity. We found that, regardless of muscle length in immobilized limbs, the mRNA of a marker for mitochondrial quantity, cytochrome c,decreased. Cytochrome c mRNA permilligram of muscle was 62 and 72% less 1 wk after fixation of thesoleus muscle in shortened and lengthened positions, respectively, thanage-matched controls. Cytochrome cmRNA per milligram wet weight was 36 and 32% less in the tibialisanterior muscle fixed for 1 wk in the shortened and lengthenedpositions, respectively, compared with age-matched controls. Recently,in the 3-untranslated region of cytochromec mRNA a novel RNA-protein interactionthat decreases in chronically stimulated rat skeletal musclewas identified.[Z. Yan, S. Salmons, Y. L. Dang, M. T. Hamilton, and F. W. Booth. Am. J. Physiol. 271 (CellPhysiol. 40): C1157- C1166,1996]. The RNA-protein interaction inthe 3-untranslated region of cytochrome c mRNA in soleus and tibialis anteriormuscles was unaffected by fixation in either shortened or lengthenedposition. We conclude that, whereas lengthening muscle during limbfixation abates the loss of total muscle protein, the percentagedecrease in cytochrome c mRNA isproportionally greater than total protein. This suggests that thedesign of countermeasures to muscle atrophy should include differentexercises to maintain total protein and mitochondria.

     

Endothelial modulation of neural sympathetic vascular tone in canine skeletal muscle

The effect of nitric oxide synthase (NOS)inhibition and endothelin-A(ET_A)-receptor blockade onneural sympathetic control of vascular tone in the gastrocnemius musclewas examined in anesthetized dogs under conditions of constant flow.Muscle perfusion pressure (MPP) was measured before and after NOSinhibition(N-nitro-L-argininemethyl ester; L-NAME) andET_A-receptor blockade [cyclo-(D-Trp-d-Asp-Pro-D-Val-Leu);BQ-123]. Zero and maximum sympathetic nerve activities wereachieved by sciatic nerve cold block and stimulation, respectively. Ingroup 1 (n = 6), MPP was measured1) before nerve cold block,2) during nerve cold block, and3) during nerve stimulation.Measurements under these conditions were repeated afterL-NAME and then BQ-123. The sameprotocol was followed in group 2 (n = 6) except that the order ofL-NAME and BQ-123 was reversed.MPP and muscle vascular resistance (MVR) increased afterL-NAME and then decreased tocontrol values after BQ-123. MVR decreased after BQ-123 alone and, withthe addition of L-NAME,increased to a level not different from that observed during thecontrol period. MVR fell during nerve cold block. This response was notaffected by administration ofL-NAME followed by BQ-123, butit was attenuated by administration of BQ-123 before L-NAME. The constrictor responseduring sympathetic nerve stimulation was enhanced byL-NAME; no further effect wasobserved with BQ-123, nor was the response affected when BQ-123 wasgiven first. These findings indicate that endothelin contributes to1) basal vascular tone in skeletalmuscle and 2) the increase inskeletal muscle vascular resistance after NOS inhibition. Finally,nitric oxide "buffers" the degree of constriction in skeletalmuscle vasculature during maximal sympathetic stimulation.

     

Exhalation of gaseous nitric oxide by rats in response to endotoxin and its absorption by the lungs

Stitt, John T., Arthur B. DuBois, James S. Douglas, andSteven G. Shimada. Exhalation of gaseous nitric oxide by rats inresponse to endotoxin and its absorption by the lungs.J. Appl. Physiol. 82(1): 305-316, 1997.Rats injected with a lipopolysaccharide endotoxin producedetectable concentrations of nitric oxide gas (NO) in the expired airwithin 60 min. The concentration of NO reaches a plateau at 3 h. Production of the NO is dose dependent onlipopolysaccharide, and at a dose of 1 mg/kg iv, lipopolysaccharide alveolar concentrations of >260 parts per billion are observed. NOsynthase inhibitors suppress this NO production in response toendotoxin. Experiments were conducted to ascertain the site of originof this NO and to measure the capacity of the lungs to absorb NO fromalveolar air. Results indicate that the endotoxin-induced NO originatesfrom within the lungs themselves and that the lungs have the capacityto absorb >60% of NO that is presented to them. Lung tissues absorb~44-47% of the NO load, blood carries away between 15 and 19%,while the remainder is exhaled in the expired air. It is proposed thatthe exhalation of NO might prove useful as an early biomarker for acutelung injury.

     

Long-term facilitation of upper airway muscle activities in vagotomized and vagally intact cats

Mateika, J. H., and R. F. Fregosi. Long-termfacilitation of upper airway muscle activities in vagotomized andvagally intact cats. J. Appl. Physiol.82(2): 419-425, 1997.The primary purpose of the presentinvestigation was to determine whether long-term facilitation (LTF) ofupper airway muscle activities occurs in vagotomized and vagally intactcats. Tidal volume and diaphragm, genioglossus, and nasal dilatormuscle activities were recorded before, during, and after one carotidsinus nerve was stimulated five times with 2-min trains of constantcurrent. Sixty minutes after stimulation, nasal dilator andgenioglossus muscle activities were significantly greater than controlin the vagotomized cats but not in the vagally intact cats. Tidalvolume recorded from the vagotomized and vagally intact cats wassignificantly greater than control during the poststimulation period.In contrast, diaphragm activities were not significantly elevated inthe poststimulation period in either group of animals. We conclude that1) LTF of genioglossus and nasaldilator muscle activities can be evoked in vagotomized cats;2) vagal mechanisms inhibit LTF inupper airway muscles; and 3) LTF canbe evoked in accessory inspiratory muscles because LTF of inspiredtidal volume was greater than LTF of diaphragm activity.

     

Nitric oxide and vasodilation in human limbs

Joyner, Michael J., and Niki M. Dietz.Nitric oxide and vasodilation in human limbs. J. Appl. Physiol. 83(6): 1785-1796, 1997.Both theskeletal muscle and skin of humans possess remarkable abilities tovasodilate. Marked vasodilation can be seen in these vascular beds inresponse to a variety of common physiological stimuli. These stimuliinclude reactive hyperemia (skin and muscle), exercise hyperemia(muscle), mental stress (muscle), and whole body heating (skin). Thephysiological mechanisms that cause vasodilation in response to thesestimuli are poorly understood, and the substance(s) responsible for itremain unclear. In this context, recent attention has been focused onthe possible contribution of nitric oxide (NO) to the regulation ofhyperemic responses in human skin and skeletal muscle. The emergingpicture is that NO is not an essential component of the dilatorresponse seen during reactive hyperemia. However, it does appear thatNO may play a modest role in exercise hyperemia. NO appears to play amajor role in the skeletal muscle vasodilation seen in response tomental stress in humans. Preliminary evidence also indicates that NO isnot essential for the normal dilator responses observed in thecutaneous circulation during body heating in humans, but this issueneeds further study. There are a number of possible mechanisms thatmight mediate NO release in humans, and the role of these mechanisms inthe various hyperemic responses is also poorly understood. The role ofaltered NO-mediated vasodilation in some disease states is alsodiscussed. Whereas NO is a potent vasodilating substance, the actionsof NO alone do not explain a variety of poorly understood vasodilatormechanisms in conscious humans. Much work remains for those interestedin the role of NO in the regulation of blood flow to the skin and skeletal muscle of humans.

     

Appendicular skeletal muscle mass: effects of age, gender, and ethnicity

Gallagher, Dympna, Marjolein Visser, Ronald E. De Meersman,Dennis Sepúlveda, Richard N. Baumgartner, Richard N. Pierson, Tamara Harris, and Steven B. Heymsfield. Appendicular skeletal muscle mass: effects of age, gender, and ethnicity. J. Appl. Physiol. 83(1): 229-239, 1997.This studytested the hypothesis that skeletal muscle mass is reduced in elderlywomen and men after adjustment first for stature and body weight. Thehypothesis was evaluated by estimating appendicular skeletal musclemass with dual-energy X-ray absorptiometry in a healthy adult cohort. Asecond purpose was to test the hypothesis that whole body⁴⁰K counting-derived total bodypotassium (TBK) is a reliable indirect measure of skeletal muscle mass.The independent effects on both appendicular skeletal muscle and TBK ofgender (n = 148 women and 136 men) andethnicity (n = 152 African-Americans and 132 Caucasians) were also explored. Main findingswere 1) for both appendicularskeletal muscle mass (total, leg, and arm) and TBK, age was anindependent determinant after adjustment first by stepwise multipleregression for stature and weight (multiple regression modelr² = ~0.60);absolute decrease with greater age in men was almost double that inwomen; significantly larger absolute amounts were observed in men andAfrican-Americans after adjustment first for stature, weight, and age;and >80% of within-gender or -ethnic group between-individualcomponent variation was explained by stature, weight, age, gender, andethnicity differences; and 2) mostof between-individual TBK variation could be explained by totalappendicular skeletal muscle(r² = 0.865),whereas age, gender, and ethnicity were small but significant additional covariates (totalr² = 0.903). Ourstudy supports the hypotheses that skeletal muscle is reduced in theelderly and that TBK provides a reasonable indirect assessment ofskeletal muscle mass. These findings provide a foundation forinvestigating skeletal muscle mass in a wide range of health-related conditions.

     

Strength, skeletal muscle composition, and enzyme activity in multiple sclerosis

Kent-Braun, J. A., A. V. Ng, M. Castro, M. W. Weiner, D. Gelinas, G. A. Dudley, and R. G. Miller. Strength, skeletal musclecomposition and enzyme activity in multiple sclerosis. J. Appl. Physiol. 83(6):1998-2004, 1997.This study examined functional, biochemical, andmorphological characteristics of skeletal muscle in nine multiplesclerosis (MS) patients and eight healthy controls in an effort toascertain whether intramuscular adaptations could account for excessivefatigue in this disease. Analyses of biopsies of the tibialis anteriormuscle showed that there were fewer type I fibers (66 ± 6 vs. 76 ± 6%), and that fibers of all types were smaller (average26%) and had lower succinic dehydrogenase (SDH; average40%) and SDH/-glycerol-phosphate dehydrogenase (GPDH) butnot GPDH activities in MS vs. control subjects, suggesting that musclein this disease is smaller and relies more on anaerobic thanaerobic-oxidative energy supply than does muscle of healthyindividuals. Maximal voluntary isometric force fordorsiflexion was associated with both average fiber cross-sectionalarea (r = 0.71, P = 0.005) and muscle fat-free cross-sectional area by magnetic resonance imaging(r = 0.80, P < 0.001). Physical activity,assessed by accelerometer, was associated with average fiber SDH/GPDH(r = 0.78, P = 0.008). There was a tendency forsymptomatic fatigue to be inversely associated with average fiber SDHactivity (r = 0.57,P = 0.068). The results of thisstudy suggest that the inherent characteristics of skeletal musclefibers per se and of skeletal muscle as a whole are altered in thedirection of disuse in MS. They also suggest that changes in skeletalmuscle in MS may significantly affect function.

     

Role of nitric oxide on diaphragmatic contractile failure in Escherichia coli endotoxemic rats

Contractile dysfunction of the respiratory muscles plays an important role in the genesis of respiratory failure during sepsis. Nitric oxide (NO), a free radical that is cytotoxic and negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by a NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to investigate whether iNOS was induced in the diaphragm of Escherichia coli endotoxemic rats and whether inhibition of iNOS induction or of NOS synthesis attenuated diaphragmatic contractile dysfunction. Rats were inoculated intravenously (IV) with 10 mg/kg of E. coli endotoxin (LPS animals) or saline (C animals). Six hours after LPS inoculation animals showed a significant increase in diaphragmatic NOS activity (L-citrulline production, P < 0.005). Inducible NOS protein was detected by Western-Blot in the diaphragms of LPS animals, while it was absent in C animals. LPS animals had a significant decrease in diaphragmatic force (P < 0.0001) measured in vitro. In LPS animals, inhibition of iNOS induction with dexamethasone (4 mg/kg IV 45 min before LPS) or inhibition of NOS activity with N(G)-methyl-L-arginine (8 mg/kg IV 90 min after LPS) prevented LPS-induced diaphragmatic contractile dysfunction. We conclude that increased NOS activity due to iNOS was involved in the genesis of diaphragmatic dysfunction observed in E. coli endotoxemic rats.     

Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles

Connor, Michael K., and David A. Hood. Effect ofmicrogravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles. J. Appl.Physiol. 84(2): 593-598, 1998.The purpose ofthis study was to examine the expression of nuclear and mitochondrialgenes in cardiac and skeletal muscle (triceps brachii) in response toshort-duration microgravity exposure. Six adult male rats were exposedto microgravity for 6 days and were compared with six ground-basedcontrol animals. We observed a significant 32% increase in heartmalate dehydrogenase (MDH) enzyme activity, which was accompanied by a62% elevation in heart MDH mRNA levels after microgravity exposure.Despite modest elevations in the mRNAs encoding subunits III, IV, andVIc as well as a 2.2-fold higher subunit IV protein content afterexposure to microgravity, heart cytochromec oxidase (CytOx) enzyme activityremained unchanged. In skeletal muscle, MDH expression was unaffectedby microgravity, but CytOx activity was significantly reduced 41% bymicrogravity, whereas subunit III, IV, and VIc mRNA levels and subunitIV protein levels were unaltered. Thus tissue-specific (i.e., heart vs.skeletal muscle) differences exist in the regulation of nuclear-encoded mitochondrial proteins in response to microgravity. In addition, theexpression of nuclear-encoded proteins such as CytOx subunit IV andexpression of MDH are differentially regulated within a tissue. Ourdata also illustrate that the heart undergoes previously unidentifiedmitochondrial adaptations in response to short-term microgravityconditions more dramatic than those evident in skeletal muscle. Furtherstudies evaluating the functional consequences of these adaptations inthe heart, as well as those designed to measure protein turnover, arewarranted in response to microgravity.

     

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.

     

Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade

Blockade or genedeletion of inducible nitric oxide synthase (iNOS) fails to fullyabrogate all the sequelae leading to the high morbidity of septicemia.An increase in substrate uptake may be necessary for the increasedproduction of nitric oxide (NO), but arginine is also a precursor forother bioactive products. Herein, we demonstrate an increase inalternate arginine products via arginine and ornithine decarboxylase inrats given lipopolysaccharide (LPS). The expression of iNOS mRNA inrenal tissue was evident 60 but not 30 min post-LPS, yet a rapiddecrease in blood pressure was obtained within 30 min that wascompletely inhibited by selective iNOS blockade. Plasma levels ofarginine and ornithine decreased by at least 30% within 60 min of LPSadministration, an effect not inhibited by the iNOS blockerL-N⁶(1-iminoethyl)lysine(L-NIL). Significant increases in plasma nitrates andcitrulline occurred only 3-4 h post-LPS, an effect blocked byL-NIL pretreatment. The intracellular composition of organsharvested 6 h post-LPS reflected tissue-specific profiles of arginineand related metabolites. Tissue arginine concentration, normally anorder of magnitude higher than in plasma, did not decrease after LPS.Pretreatment with L-NIL had a significant impact on thedisposition of tissue arginine that was organ specific. These datademonstrate changes in arginine metabolism before and after de novoiNOS activity. Selective blockade of iNOS did not prevent uptake andcan deregulate the production of other bioactive arginine metabolites.

     

Effects of transverse fiber stiffness and central tendon on displacement and shape of a simple diaphragm model

Boriek, Aladin M., and Joseph R. Rodarte. Effects oftransverse fiber stiffness and central tendon on displacement and shapeof a simple diaphragm model. J. Appl. Physiol. 82(5): 1626-1636, 1997.Our previous experimental results (A. M. Boriek, S. Lui, and J. R. Rodarte. J. Appl. Physiol. 75:527-533, 1993 and A. M. Boriek, T. A. Wilson, and J. R. Rodarte.J. Appl. Physiol. 76: 223-229, 1994) showed that1) costal diaphragm shape is similar at functional residualcapacity and end inspiration regardless of whether the diaphragm muscleshortens actively (increased tension) or passively (decreased tension);2) diaphragmatic muscle length changes minimally in thedirection transverse to the muscle fibers, suggesting the diaphragm maybe inextensible in that direction; and 3) the central tendon isnot stretched by physiological stresses. A two-dimensional orthotropicmaterial has two different stiffnesses in orthogonal directions. In theplane tangent to the muscle surface, these directions are along thefibers and transverse to the fibers. We wondered whether orthotropicmaterial properties in the muscular region of the diaphragm andinextensibility of the central tendon might contribute to the constancyof diaphragm shape. Therefore, in the present study, we examined theeffects of stiffness transverse to muscle fibers and inextensibility ofthe central tendon on diaphragmatic displacement and shape. Finiteelement hemispherical models of the diaphragm were developed by usingpressurized isotropic and orthotropic membranes with a wide range ofstiffness ratios. We also tested heterogeneous models, in which themuscle sheet was an orthotropic material, having transverse fiberstiffness greater than that along the fibers, with the central tendonbeing an inextensible isotropic cap. These models revealed thatincreased transverse stiffness limits the shape change of thediaphragm. Furthermore, an inextensible cap simulating the centraltendon dramatically limits the change in shape as well as the membrane displacement in response to pressure. These findings provide a plausible mechanism by which the diaphragm maintains similar shapes despite different physiological loads. This study suggests that changesof diaphragm shape are restricted because the central tendon isessentially inextensible and stiffness in the direction transverse tothe muscle fibers is greater than stiffness along the fibers.

     

Nitric oxide derived from sympathetic nerves regulates airway responsiveness to histamine in guinea pigs

Matsumoto, Koichiro, Hisamichi Aizawa, Shohei Takata,Hiromasa Inoue, Naotsugu Takahashi, and Nobuyuki Hara.Nitric oxide derived from sympathetic nerves regulates airwayresponsiveness to histamine in guinea pigs. J. Appl.Physiol. 83(5): 1432-1437, 1997.Nitric oxide(NO), which can be derived from the nervous system or the epithelium ofthe airway, may modulate airway responsiveness. We investigated how NOderived from the airway nervous system would affect the airwayresponsiveness to histamine and acetylcholine in mechanicallyventilated guinea pigs. An NO synthase inhibitor N^G-nitro-L-argininemethyl ester (L-NAME) (1 mmol/kgip) significantly enhanced airway responsiveness to histamine but notto acetylcholine. Its enantiomerD-NAME (1 mmol/kg ip), incontrast, had no effect. TheL-NAME-induced airwayhyperresponsiveness was still observed in animals pretreated withpropranolol (1 mg/kg iv) and atropine (1 mg/kg iv). Pretreatment withthe ganglionic blocker hexamethonium (2 mg/kg iv) completely abolishedenhancing effect of L-NAME on airway responsiveness. Bilateral cervical vagotomy did not alter theL-NAME-induced airwayhyperresponsiveness, whereas sympathetic stellatectomy completelyabolished it. Results suggest that NO that was presumably derived fromthe sympathetic nervous system regulates airway responsiveness tohistamine in guinea pigs.

     

Inducible nitric-oxide synthase and NO donor induce insulin receptor substrate-1 degradation in skeletal muscle cells

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.