Force generation,but not myosin ATPase activity,declines with age in rat muscle fibers

We tested the hypothesis thatage-associated decline in muscle function is related to a change inmyosin ATPase activity. Single, glycerinated semimembranosus fibersfrom young (8-12 mo) and aged (32-37 mo) Fischer 344 × Brown Norway male rats were analyzed simultaneously for force andmyosin ATPase activity over a range of Ca²⁺ concentrations.Maximal force generation was ~20% lower in fibers from aged animals(P = 0.02), but myosin ATPase activity was not different between fibers from young and aged rats: 686 ± 46 (n = 30) and 697 ± 46 µM/s (n = 33) (P = 0.89). The apparent rate constant for thedissociation of strong-binding myosin from actin was calculated to be~30% greater in fibers from aged animals (P = 0.03),indicating that the lower force produced by fibers from aged animals isdue to a greater flux of myosin heads from the strong-binding state tothe weak-binding state during contraction. This is in agreement withour previous electron paramagnetic resonance experiments that showed areduced fraction of myosin heads in the strong-binding state during amaximal isometric contraction in fibers from older rats.

     

Glutathione reverses early effects of glycation on myosin function

Nonenzymatic glycosylation (glycation) has been recognized as an important posttranslational modification underlying alterations of structure and function of extracellular proteins during aging and diabetes. Intracellular proteins may also be affected by this modification, and glycation has been suggested to contribute to aging-related impairment in skeletal muscle function. Glycation is the chemical reaction of reducing sugars with primary amino groups resulting in the formation of irreversible advanced glycation end products. Glutathione is an abundant tripeptide in skeletal muscle. To understand the effect of glutathione on glycated myosin function, we used a single-fiber in vitro motility assay in which myosin is extracted from a single muscle fiber segment to propel fluorescent-labeled actin filaments. Myosin function responded to glucose exposure in a dose-dependent manner, i.e., motility speeds were reduced by 10, 34, and 90% of preincubation values after 30-min exposure to 1, 3, and 6 mM glucose, respectively. The 30-min 6 mM glucose incubation was followed by a 20-min 10 mM glutathione incubation. Glutathione treatment restored motility (0.98 ± 0.06 µm/s, n = 3; P < 0.001) after glucose exposure (0.10 ± 0.07 µm/s, n = 3), close to preincubation levels (1.12 ± 0.06 µm/s, n = 3). It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function. in vitro motility; skeletal muscle fibers; speed of contraction     

Length-dependent modulation of smooth muscle activation: effects of agonist, cytochalasin, and temperature

We tested thehypothesis that mechanical strain modulates agonist sensitivity ofsmooth muscle by measuring myosin phosphorylation and contractile forcein bovine tracheal smooth muscle activated by various concentrations ofthe muscarinic receptor agonist carbachol and at various musclelengths. Increasing carbachol concentration by 10,000-fold did notrestore myosin phosphorylation levels at shorter muscle lengths to thelevel at optimal length(L_o). Maximum levels of myosin phosphorylation induced by carbachol at 0.6, 0.8, and1.0 L_o weresimilar but became lower at <0.6L_o. Cytochalasin D significantly attenuated carbachol-induced contraction by 54%. Inaddition, cytochalasin D treatment induced a parallel downward shift inthe length-myosin phosphorylation relation. Lowering temperature from37 to 23°C did not significantly change the length dependencies ofcarbachol-induced active force and myosin phosphorylation. Theseresults have led us to conclude that1) agonist sensitivity and maximumlevel of activation (as measured by myosin phosphorylation) are targetsof length-dependent modulation, 2)actin filaments involved in contraction and length-dependent modulationare distinct in sensitivity to cytochalasin D, and3) length-dependent modulation isrelatively temperature insensitive.

     

Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity

Isolated single smoothmuscle cells (SMCs) from different regions of the rabbit stomach wereused to determine a possible correlation between unloaded shorteningvelocity and smooth muscle (SM) myosin heavy chain (MHC) S1 headisoform composition (SMA, no head insert; SMB, with head insert).-Toxin-permeabilized isolated single cells were maximally activatedto measure unloaded shortening velocity and subsequently used in anRT-PCR reaction to determine the SMA/SMB content of the same cell. SMMHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 ± 7.3% SMB; n = 16); cells from the antrum express primarilySMB (94.9 ± 1.0% SMB; n = 16). Mean fundic cellunloaded shortening velocity was 0.014 ± 0.002 cell lengths/scompared with 0.036 ± 0.002 for the antrum cells. Unloadedshortening velocity in these cells was significantly correlated withtheir percent SMB expression (r² = 0.58).Resting cell length does not correlate with the percent SMB expression(n = 32 cells). Previously published assays of purifiedor expressed SMA and SMB heavy meromyosin show a twofold difference inactin filament sliding speed in in vitro motility assays. Extrapolationof our data to 0-100% SMB would give a 10-fold range ofshortening velocity, which is closer to the ~20-fold range reportedfrom various SM tissues. This suggests that mechanisms in addition tothe MHC S1 head isoforms regulate shortening velocity.

     

Modulation of myosin isoform expression by mechanical loading: role of stimulation frequency

Caiozzo, Vincent J., Michael J. Baker, and Kenneth M. Baldwin. Modulation of myosin isoform expression by mechanical loading: role of stimulation frequency. J. Appl.Physiol. 82(1): 211-218, 1997.This study testedthe hypothesis that mechanical loading, not stimulation frequency perse, plays a key role in determining the plasticity of myosin heavychain (MHC) protein isoform expression in muscle undergoing resistancetraining. Female Sprague-Dawley rats were randomly assigned toresistance-training programs that employed active1) shortening(n = 7) or2) lengthening contractions(n = 8). The medial gastrocnemius (MG)muscles in each group trained under loading conditions thatapproximated 90-95% of maximum isometric tetanictension but were stimulated at frequencies of 100 and~25 Hz, respectively. Lengthening and shortening contractions wereproduced by using a Cambridge ergometer system. The MG muscles trainedevery other day, performing a total of 16 training sessions. Bothtraining programs produced significant (P < 0.01) and similar reductions inthe fast type IIB MHC protein isoform in the white MG muscle, reducingits relative content to ~50% of the total MHC protein isoform pool.These changes were accompanied by increases in the relative content ofthe fast type IIX MHC protein isoform that were of similar magnitudefor both groups. The results of this study clearly demonstrate thatstimulation frequency does not play a key role in modulating MHCisoform alterations that result from high-resistance training.

     

Comparisons of two-, three-, and four-compartment models of body composition analysis in men and women

This study compared the traditionaltwo-compartment (fat mass or FM; fat free mass or FFM)hydrodensitometric method of body composition measurement, which isbased on body density, with three (FM, total body water or TBW, fatfree dry mass)- and four (FM, TBW, bone mineral mass or BMM,residual)-compartment models in highly trained men(n = 12), sedentary men(n = 12), highly trained women(n = 12), and sedentary women(n = 12). The means andvariances for the relative body fat (%BF) differences between the two-and three-compartment models [2.2 ± 1.6 (SD) % BF;n = 48] were significantlygreater (P  0.02) than those between the three- and four-compartment models (0.2 ± 0.3% BF;n = 48) for all four groups. Thethree-compartment model is more valid than the two-compartmenthydrodensitometric model because it controls for biological variabilityin TBW, but additional control for interindividual variability in BMMvia the four-compartment model achieves little extra accuracy. Thecombined group (n = 48) exhibited greater (P < 0.001) FFM densities(1.1075 ± 0.0049 g/cm³) thanthe hydrodensitometric assumption of 1.1000 g/cm³, which is based on analysesof three male cadavers aged 25, 35, and 46 yr. This was primarilybecause their FFM hydration (72.4 ± 1.1%;n = 48) was lower(P  0.001) than thehydrodensitometric assumption of 73.72%.

     

Influence of diet and exercise on skeletal muscle and visceral adipose tissue in men

Ross, Robert, John Rissanen, Heather Pedwell, JenniferClifford, and Peter Shragge. Influence of diet and exercise onskeletal muscle and visceral adipose tissue in men. J. Appl. Physiol. 81(6): 2445-2455, 1996.Theeffects of diet only (DO) and diet combined with either aerobic (DA) orresistance (DR) exercise on subcutaneous adipose tissue (SAT), visceraladipose tissue (VAT), lean tissue (LT), and skeletal muscle (SM) tissue were evaluated in 33 obese men (DO, n = 11; DA, n = 11; DR,n = 11). All tissues were measured byusing a whole body multislice magnetic resonance imaging (MRI) model.Within each group, significant reductions were observed for bodyweight, SAT, and VAT (P < 0.05). Thereductions in body weight (~10%) and SAT (~25%) and VAT volume (~35%) were not different between groups(P > 0.05). For alltreatments, the relative reduction in VAT was greater than in SAT(P < 0.05). For the DA and DR groupsonly, the reduction in abdominal SAT (~27%) was greater(P < 0.05) than thatobserved for the gluteal-femoral region (~20%). Conversely, thereduction in VAT was uniform throughout the abdomen regardless oftreatment (P > 0.05). MRI-LT andMRI-SM decreased both in the upper and lower body regions for the DO group alone (P < 0.05). PeakO₂ uptake (liters) wassignificantly improved (~14%) in the DA group as was muscularstrength (~20%) in the DR group (P < 0.01). These findings indicate that DA and DR result in a greaterpreservation of MRI-SM, mobilization of SAT from the abdominal region,by comparison with the gluteal-femoral region, and improved functionalcapacity when compared with DO in obese men.

     

The converter domain modulates kinetic properties of Drosophila myosin

Recently the converter domain, anintegral part of the "mechanical element" common to all molecularmotors, was proposed to modulate the kinetic properties ofDrosophila chimeric myosin isoforms. Here we investigatedthe molecular basis of actin filament velocity(V_actin) changes previously observed with thechimeric EMB-IC and IFI-EC myosin proteins [the embryonic body wallmuscle (EMB) and indirect flight muscle isoforms (IFI) with geneticsubstitution of the IFI and EMB converter domains, respectively]. Inthe laser trap assay the IFI and IFI-EC myosins generate the sameunitary step displacement (IFI = 7.3 ± 1.0 nm, IFI-EC = 5.8 ± 0.9 nm; means ± SE). Thus converter-mediateddifferences in the kinetics of strong actin-myosin binding, rather thanthe mechanical capabilities of the protein, must account for theobserved V_actin values. Basal andactin-activated ATPase assays and skinned fiber mechanical experimentsdefinitively support a role for the converter domain in modulating thekinetic properties of the myosin protein. We propose that the converterdomain kinetically couples the P_i and ADP release stepsthat occur during the cross-bridge cycle.

     

Myosin molecular motor dysfunction in dystrophic mouse diaphragm

Cross-bridge properties and myosin heavy chain (MHC) compositionwere investigated in isolated diaphragm from 6-mo-old control (n = 12) andmdx(n = 12) mice. Compared with control,peak tetanic tension fell by 50% inmdx mice(P < 0.001). The total number ofcross bridges per square millimeter(×10⁹), the elementaryforce per cross bridge, and the peak mechanical efficiency were lowerin mdx than in control mice (eachP < 0.001). The duration of thecycle and the rate constant for cross-bridge detachment weresignificantly lower in mdx than incontrol mice. In the overall population, there was a linearrelationship between peak tetanic tension and either total number ofcross bridges per square millimeter or elementary force per crossbridge (r = 0.996 andr = 0.667, respectively, eachP < 0.001). Themdx mice presented a higher proportionof type IIA MHC (P < 0.001) thancontrol mice and a reduction in type IIX MHC(P < 0.001) and slowmyosin isoforms (P < 0.01) comparedwith control mice. We concluded that, inmdx mice, impaired diaphragm strengthwas associated with qualitative and quantitative changes in myosin molecular motors. It is proposed that reduced force generated per crossbridge contributed to diaphragm weakness inmdx mice.

     

Volume capacity and contraction control of the seal spleen

Cabanac, Arnaud, Lars P. Folkow, and Arnoldus Schytte Blix.Volume capacity and contraction control of the seal spleen. J. Appl. Physiol. 82(6):1989-1994, 1997.Volume changes in the spleens of hooded seals(Cystophora cristata) and harp seals(Phoca groenlandica) were measuredplethysmographically in vitro in response to epinephrine,norepinephrine, isoprenaline, phentolamine, and acetylcholine. Dilated spleens contracted forcefullywithin 1-3 min of -adrenoceptor activation with 1.0-5.0µg epinephrine/kg body mass, whereas stimulation of -adrenoceptorsand cholinergic receptors had little effect. The mass of dilated hoodedseal spleens corresponded to 2-4%(n = 7) of body mass, with volume (V;ml) relating to body mass (M; kg) as follows: V = 12.0M + 910 (r² = 0.96, n = 4). Thus the spleen of a 250-kghooded seal maximally expels 3.9 liters, or 13%, of its estimatedtotal blood volume. Average hematocrit in splenic venous outflow fromdilated spleens was 90 ± 3% (n = 3) in hooded seals and 85% (n = 2) inharp seals. From these data we have estimated that the aerobic divinglimit of a 250-kg hooded seal increases only 105 s, at the most, if complete emptying of the spleen occurs during diving, while the corresponding estimate for a 112-kg harp seal is 80 s.

     

Effect of leucine metabolite beta -hydroxy-beta -methylbutyrate on muscle metabolism during resistance-exercise training

Nissen, S., R. Sharp, M. Ray, J. A. Rathmacher, D. Rice, J. C. Fuller, Jr., A. S. Connelly, and N. Abumrad. Effect of leucinemetabolite -hydroxy--methylbutyrate on muscle metabolism duringresistance-exercise training. J. Appl.Physiol. 81(5): 2095-2104, 1996.The effects ofdietary supplementation with the leucine metabolite-hydroxy--methylbutyrate (HMB) were studied in two experiments.In study 1, subjects(n = 41) were randomized among threelevels of HMB supplementation (0, 1.5 or 3.0 g HMB/day) and two proteinlevels (normal, 117 g/day, or high, 175 g/day) and weight lifted for1.5 h 3 days/wk for 3 wk. In study 2,subjects (n = 28) were fed either 0 or3.0 g HMB/day and weight lifted for 2-3 h 6 days/wk for 7 wk. Instudy 1, HMB significantly decreased the exercise-induced rise in muscle proteolysis as measured by urine3-methylhistidine during the first 2 wk of exercise (linear decrease,P < 0.04). Plasma creatinephosphokinase was also decreased with HMB supplementation(week 3, linear decrease,P < 0.05). Weight lifted wasincreased by HMB supplementation when compared with the unsupplementedsubjects during each week of the study (linear increase,P < 0.02). In study2, fat-free mass was significantly increased inHMB-supplemented subjects compared with the unsupplemented group at 2 and 4-6 wk of the study (P < 0.05). In conclusion, supplementation with either 1.5 or 3 g HMB/daycan partly prevent exercise-induced proteolysis and/or muscledamage and result in larger gains in muscle function associated withresistance training.

     

Endurance training affects myosin heavy chain phenotype in regenerating fast-twitch muscle

Bigard, Xavier A., Chantal Janmot, Danièle Merino,Françoise Lienhard, Yannick C. Guezennec, and Anne D'Albis.Endurance training affects myosin heavy chain phenotype inregenerating fast-twitch muscle. J. Appl.Physiol. 81(6): 2658-2665, 1996.The aim of thisstudy was to analyze the effects of treadmill training (2 h/day, 5 days/wk, 30 m/min, 7% grade for 5 wk) on the expression of myosinheavy chain (MHC) isoforms during and after regeneration of afast-twitch white muscle [extensor digitorum longus (EDL)]. Male Wistar rats were randomly assigned to a sedentary(n = 10) or an endurance-trained (ET;n = 10) group. EDL muscle degeneration and regeneration were induced by two subcutaneous injections of a snaketoxin. Five days after induction of muscle injury, animals were trainedover a 5-wk period. It was verified that ~40 days after venomtreatment, central nuclei were present in the treated EDL muscles fromsedentary and ET rats. The changes in the expression of MHCs in EDLmuscles were detected by using a combination of biochemical andimmunocytochemical approaches. Compared with contralateral nondegenerated muscles, relative concentrations of types I, IIa, andIIx MHC isoforms in ET rats were greater in regenerated EDL muscles(146%, P < 0.05; 76%,P < 0.01; 87%,P < 0.01, respectively). Their elevation corresponded to a decreasein the relative concentration of type IIb MHC (36%,P < 0.01). Although type I accountedfor only 3.2% of total myosin in regenerated muscles from the ETgroup, the cytochemical analysis showed that the proportion of positive staining with the slow MHC antibody was markedly greater in regenerated muscles than in contralateral ones. Collectively, these results demonstrate that the regenerated EDL muscle is sensitive to endurance training and suggest that the training-induced shift in MHC isoforms observed in these muscles resulted from an additive effect of regeneration and repeated exercise.

     

Morphological, Biochemical and Immunohistochemical Studies on Heart Development in Cardiac Mutant Axolotls, Ambystoma mexicanum

SYNOPSIS. A naturally-occurring genetic mutation, designatedc for "cardiac lethal" in axolotls, Ambystoma mexicanum, isproving to be a useful model for studying myofibrillogenesisin differentiating heart cells. In this paper I describe morphological,biochemical and immunofluorescence studies which compare thecontractile proteins in normal and mutant hearts. In addition,morphological studies on anterior endoderm, an important heartinductor tissue in salamanders, are reviewed. Detailed electronmicroscopic studies show that normal heart myocytes containnumerous well-organized myofibrils. Although mutant heart cellscontain a few myosin and actin filaments, there are no organizedmyofibrils. Instead, amorphous proteinaceous collections areprominent in the peripheral cytoplasm of the cell where myofibrilswould be expected to first form. SDS-polyacrylamide gel electrophoresisshows that actin is present in almost normal amounts in mutanthearts, myosin heavy chain is reduced and tropomyosin is virtuallyabsent. Immunofluorescence studies reveal that myosin, -actininand tropomyosin are located prominently in theorganized myofibrilsof normal heart cells. In mutant hearts myosin is localizedalmost exclusively in the amorphous collections at the cellperipheries, -actinin also is distributed mainly in the peripheralcell cytoplasm. There is almost no staining for tropomyosin.Heavy meromyosin (HMM) binding experiments demonstrate thatthe actin in mutant heart cells is contained within the amorphouscollections in a non-filamentous state and the addition of HMMcauses its polymerization into filaments. In view of these findings,we undertook studies to determine whether there might be a causalrelationship between theabsence of tropomyosin in mutants andthe failure of actin to form into filaments. Our results indeedshow that addition of tropomyosin to glycerinated mutant heartsor homogenates of mutant hearts causes the amorphous actin toform into filaments. Thus, this single gene mutation resultsin mutant heart cells having reduced, but significant, amountsof myosin and actin, even though non-filamentous, and substantialamounts of -actinin. There is almost no tropomyosin. It is impliedthat the drastic reduction of tropomyosin in mutant cells issomehow related to the failure of normal myofilament formation,which in turn would seem to be an essential step in the normalorganization of myofibrils.     

Angiogenesis in bronchial circulatory system after unilateral pulmonary artery obstruction

Charan, Nirmal B., and Paula Carvalho. Angiogenesis inbronchial circulatory system after unilateral pulmonary artery obstruction. J. Appl. Physiol. 82(1):284-291, 1997.We studied the effects of left pulmonary artery(LPA) ligation on the bronchial circulatory system (BCS) by using asheep model. LPA was ligated in the newborn lambs soon after birth(n = 8), and when the sheep were ~3yr of age anatomic studies revealed marked angiogenesis in BCS.Bronchial blood flow and cardiac output were studied by placing flowprobes around the bronchial and pulmonary arteries in four adult sheep.After LPA ligation, bronchial blood flow increased from 35 ± 6 to134 ± 42 ml/min in ~3 wk (P < 0.05). We also studied gas-exchange functions of BCS ~3 yr after the ligation of LPA in newborn lambs (n = 4) and used a control group (n = 12)in which LPA was ligated acutely. In the left lung,O₂ uptake after acute ligation was16 ± 3 ml/min and was similar to the chronic model, whereasCO₂ output in the control group was 27 ± 3 ml/min compared with 79 ± 12 ml/min in the chronic preparation (P < 0.05).We conclude that LPA ligation causes marked angiogenesis in BCS that iscapable of performing some gas-exchange functions.

     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.

     

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.

     

Mechanism of mosaic attenuation of the lungs on computed tomography in induced bronchospasm

The purpose of this study was to investigatewhether hypoxic pulmonary vasoconstriction is the major determinant ofthe computed tomography (CT) pattern of mosaic attenuation in asthmaticpatients with induced bronchoconstriction. Thin-section CT wasperformed at suspended full inspiration immediately and 30 min aftermethacholine bronchoprovocation in 22 asthmatic subjects, who wererandomly assigned to breathe room air (group A,n = 8), oxygen via nasal prongs at 5 l/min (group B,n = 8), and oxygen via face mask at 12 l/min (group C,n = 6). CT changes were quantified interms of global lung density and density in hypodense and hyperdense areas. Lung parenchymal density increases were greatest ingroup C and greater ingroup B than in groupA, globally (P = 0.03) and in hypodense regions (P = 0.01).On bivariate analysis, the only change in cross-sectional area wasrelated to change in global density. In hypodense regions, densitychange was related both to reduction in cross-sectional area(P < 0.0005) and to oxygen administration (P = 0.01). Aftercorrection for changes in global lung density, only oxygen wasindependently related to density increase in hypodense areas(P = 0.02). In inducedbronchoconstriction, the CT appearance of mosaic attenuation can belargely ascribed to hypoxic vasoconstriction rather than to changes inlung inflation.     

Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs

Ventilation during ischemia attenuatesischemia-reperfusion lung injury, but the mechanism is unknown.Increasing tissue cyclic nucleotide levels has been shown to attenuatelung ischemia-reperfusion injury. We hypothesized thatventilation prevented increased pulmonary vascular permeability duringischemia by increasing lung cyclic nucleotide concentrations.To test this hypothesis, we measured vascular permeability and cGMP andcAMP concentrations in ischemic (75 min) sheep lungs that wereventilated (12 ml/kg tidal volume) or statically inflated with the samepositive end-expiratory pressure (5 Torr). The reflection coefficientfor albumin (_alb) was 0.54 ± 0.07 and 0.74 ± 0.02 (SE) in nonventilated and ventilatedlungs, respectively (n = 5, P < 0.05). Filtration coefficientsand capillary blood gas tensions were not different. The effect ofventilation was not mediated by cyclic compression of alveolarcapillaries, because negative-pressure ventilation(n = 4) also was protective (_alb = 0.78 ± 0.09). Thefinal cGMP concentration was less in nonventilated than in ventilatedlungs (0.02 ± 0.02 and 0.49 ± 0.18 nmol/g blood-free dry wt,respectively, n = 5, P < 0.05). cAMP concentrations werenot different between groups or over time. Sodium nitroprussideincreased cGMP (1.97 ± 0.35 nmol/g blood-free dry wt) and_alb (0.81 ± 0.09) innonventilated lungs (n = 5, P < 0.05). Isoproterenol increasedcAMP in nonventilated lungs (n = 4, P < 0.05) but had no effect on_alb. The nitric oxide synthaseinhibitor N^G-nitro-L-arginine methylester had no effect on lung cGMP (n = 9) or _alb(n = 16) in ventilated lungs but didincrease pulmonary vascular resistance threefold(P < 0.05) in perfused sheep lungs (n = 3). These results suggest thatventilation during ischemia prevented an increase in pulmonaryvascular protein permeability, possibly through maintenance of lungcGMP by a nitric oxide-independent mechanism.

     

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3 M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤ 1 M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3 M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, V_max, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3 M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3 M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.