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(The FASEB Journal. 2001;15:1764-1770.)
© 2001 FASEB

Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle

CHELLIAH R. RICHMONDS* and HENRY J. KAMINSKI{dagger}1

Departments of
* Neurology and
{dagger} Neurosciences, Case Western Reserve University School of Medicine, Louis Stokes Cleveland Veterans Affairs Medical Center, University Hospitals of Cleveland, Cleveland, Ohio 44106, USA

1Correspondence: Department of Neurology, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH, 44106, USA. E-mail: hjk3{at}po.cwru.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Extraocular muscles (EOMs) are specialized skeletal muscles that are constantly active, generate low levels of force for cross sectional area, have rapid contractile speeds, and are highly fatigue resistant. The neuronal isoform of nitric oxide synthase (nNOS) is concentrated at the sarcolemma of fast-twitch muscles fibers, and nitric oxide (NO) modulates contractility. This study evaluated nNOS expression in EOM and the effect of NO modulation on lateral rectus muscle’s contractility. nNOS activity was highest in EOM compared with diaphragm, extensor digitorum longus, and soleus. Neuronal NOS was concentrated to the sarcolemma of orbital and global singly innervated fibers, but not evident in the multi-innervated fibers. The NG-nitro-L-arginine methyl ester (L-NAME, a NOS inhibitor), increased submaximal tetanic and peak twitch forces. The NO donors S-nitroso-N-acetylcysteine (SNAC) and spermineNONOate reduced submaximal tetanic and peak twitch forces. The effect of NO on the contractile force of lateral rectus muscle is greater than previously observed on other skeletal muscle. NO appears more important in modulating contraction of EOM compared with other skeletal muscles, which could be important for the EOM’s specialized role in generation of eye movements.—Richmonds, C. R., Kaminski, H. J. Nitric oxide synthase expression and effects of nitric oxide modulation on contractility of rat extraocular muscle.


Key Words: extraocular muscle • nitric oxide • muscle contractility • neuronal nitric oxide synthase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EXTRAOCULAR MUSCLES (EOMS) are adapted for their specialized role in generation of eye movements, which range from extremely rapid changes of gaze, to slow, steady pursuit of objects of interest, and maintaining fixation in a single position. Each eye is rotated by six EOMs: four rectus (superior, inferior, medial, and lateral) muscles and two oblique (superior and inferior) muscles. The individual muscles function as yoked pairs within fine specifications to maintain the position of the visual axes. Given the requirements placed on these muscles, physiological, structural, and molecular characteristics that differ from other skeletal muscles are expected (1 2 3) . Previous investigators have identified that EOMs have rapid contractile speeds, high fatigue resistance, and generate low levels of force compared with other skeletal muscles (2 , 4) .

The EOMs are anatomically divided. A global region lies adjacent to the eye and the orbital region is next to the bony orbit. The global layer inserts directly on the eye, whereas the orbital layer was appreciated recently to insert not on the globe, as always thought, but on a fibroelastic tissue pulley system within the orbit (5) . At least for the rectus muscles, the global layer serves to rotate the globe whereas the orbital layer inserts on its pulley to position it linearly and thus influence the EOM’s rotational axis. This arrangement appears to account for various aspects of ocular dynamics and kinematics.

The traditional classification schemes used for skeletal muscle fibers cannot be applied to EOM (Table 1 ) (3 , 6) . EOM fibers are best categorized into six distinct fiber types based on anatomic location, innervational pattern, and mitochondrial/oxidative enzyme contents. Eighty percent of EOM fibers are singly innervated fibers (SIFs), similar to those typical of other skeletal muscles. The orbital region contains a specific type of SIF, whereas the global region contains three types of SIFs. The orbital SIF fiber has a high oxidative capacity and is similar to typical type 2A fibers, but expresses embryonic/neonatal, an EOM-specific, as well as type 2A myosin isoforms (7 , 8) . The global SIFs were categorized by Spencer and Porter (3) into three distinct fiber types. The global red SIF expresses only the 2A myosin isoform and in that sense is similar to type 2A fibers of other skeletal muscle, but has a higher mitochondrial content. The global intermediate SIF expresses the 2B myosin isoform and moderate levels of oxidative and anaerobic enzymes, whereas the global pale SIF is most similar to type 2B fibers with few mitochondria and a fast myofibrillar ATPase.


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  		Table 1. NADPH diaphorase activity of EOM fiber types

The remainder of EOM fibers are multiply innervated fibers (MIFs), which are rarely found in other mammalian skeletal muscles. The global MIFs contract in a graded or ‘tonic’ fashion; the orbital MIFs can undergo graded contractions at their ends, but have twitch properties at their center (1 , 2 , 9) . The role of each of the fiber types in generation of eye movements is not known, and current theories suggest that each fiber contributes to all movements.

Nitric oxide (NO) is a free radical gas generated by nitric oxide synthases (NOS). In skeletal muscle, the neuronal isoform is the predominant form expressed as a splice variant from that found in the brain and is concentrated to the sarcolemma of fast-twitch fibers (10 , 11) . Since the majority of EOM fibers are similar to fast-twitch fibers, EOM would be expected to have a high percentage of neuronal NOS (nNOS) -containing fibers. Endothelial NOS is abundant in skeletal muscle vasculature, but otherwise is present only at low levels in fast- and slow-twitch skeletal muscle fibers, colocalizing with mitochondrial markers (12) . The inducible NOS isoform is present at low levels in rodent and human muscles, and its expression is induced by inflammatory conditions (13 , 14) .

EOM contracts against a relatively small mechanical load consisting of the globe and connective tissue, and it is not surprising that studies of a variety of species find that EOM generates a low level of total force (15 16 17) . EOM fibers across species are smaller than those of other skeletal muscle, with diameters of the orbital MIFs averaging 20 µm and the largest global SIF 46 µm (3 , 4) . This is in contrast to limb and diaphragm fibers, which have mean diameters ranging from 35 to 75 µm (18) . However, even when normalized for their small size, EOM fibers generate low levels of force (4 , 16) .

NO reduces muscle contractile force and has a short diffusion range estimated in tissue to be at most 50 µm (14 , 19) . Because EOM fibers are small, the influence of NO on contractility may be more prominent than on other muscles with NO affecting the fiber expressing nNOS, but also neighboring fibers, and offers a mechanism by which EOM force generation may be reduced. In this investigation we characterize nNOS expression in EOM and the influence of NO on EOM force generation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Dissection and tissue preparation
Adult female Lewis rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). The rats were anesthetized with intraperitoneal injections of sodium pentobarbital (40 mg/kg) and killed by cervical dislocation. For nNOS catalytic assays, all EOMs, diaphragm, extensor digitorum longus (EDL), and soleus muscles were removed and frozen in liquid N2. For histological studies, only EOMs were processed for cryostat sectioning.

For contractile studies, one lateral rectus muscle was used for each experiment. The lateral rectus was removed by lateral orbitotomy after removal of the brain. The posterior orbital processes of the frontal and zygomatic bones were cut. The eyelid was detached and the tissues surrounding the eye were removed. The lateral rectus muscle was identified and part of the sclera on either side of the muscle attachment was cut; the muscle was separated with its attachment to the sphenoid bone. The muscle was kept moist with 37°C oxygenated saline. The physiological saline solution was composed of (mM): 135 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4,1 NaH2PO4, 15 NaHCO3, and 11 glucose with pH adjusted to pH 7.4. The saline was maintained at 37°C and oxygenated with 95% O2 and 5% CO2. Incisions were made around the attachment of the muscles to the orbital rim. This separated and isolated all the EOMs with their attachments to the sphenoid bone on one end and the eye on the other. The EOMs, eyeball, and part of the sphenoid bone were transferred to a tissue bath containing physiological saline solution, maintained at 37°C, and oxygenated with 95% O2 and 5% CO2. The eye, all other muscles, and connective tissues (except the lateral rectus) were trimmed, leaving the lateral rectus muscle with portions of sclera on one end and sphenoid bone attached to the other end. The muscle was placed vertically in a double-jacketed, tissue–organ bath containing an oxygenated, physiological saline solution at 37°C. The muscle was placed between a pair of platinum electrodes (Radnoti Glass Technology, Monrovia, CA) and stimulated by an electrical field generated between these electrodes. The sphenoid attachment was tied by silk suture (6/0) to an immobile hook at the lower end of the electrode. The other end with the scleral part was attached to an isometric transducer (Radnoti Glass Technology). The transducer was connected to a computer through an Axon Instruments Digidata 1200 series A/D interface for recording the contractile force produced during electrical stimulations.

nNOS catalytic activity
Neuronal NOS activity was determined by measuring the conversion of 3H arginine to 3H citrulline at 23 ± 1°C, as described previously (20 , 21) . Muscles were homogenized on ice in 25 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA. The homogenate was assayed using a kit produced by Stratagene (La Jolla, CA) that specifically measures neuronal nNOS activity. The isolated 3H citrulline was quantitated by liquid scintillation spectroscopy. Protein concentrations of the extracts were determined using the Bio-Rad protein assay (Hercules, CA). Activity was expressed as picamoles of citrulline conversion min-1 mg-1 of protein (20) . Each sample was measured in triplicate and results combined for analysis from three animals.

NADPH diaphorase histochemistry
Using only EOM, nNOS expression was localized by NADPH diaphorase histochemistry (22) and EOM fibers were classified into the six types described by Spencer and Porter (3) . NADPH diaphorase staining is a marker of nNOS and was performed by the method of Dawson et al. (23) with modifications (24) . Serial 8 µm-thick cryostat sections were fixed at 4°C for 10 min in 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. After three rinses in PBS for 10 min, the sections were incubated in 0.2% Triton X-100 for 10 min at 37°C. The reaction was performed in a dark, humidified chamber at 37°C for 40 min in 0.2% Triton X-100, 0.1 mM NADPH, and 0.16 mg ml-1 nitro blue tetrazolium chloride. The reaction was terminated by a water wash and the sections were mounted with Immu-mount (Shandon, Pittsburgh, PA). Intense sarcolemmal staining that rings the fiber readily identifies positive fibers. A modified trichrome staining procedure was performed on serial sections to identify EOM fiber types (25) . Only sections from the midbelly of the muscle were used to correlate fiber type and NADPH diaphorase staining. Six EOM fiber types are readily differentiated by the trichrome stain and anatomic location (Table 1) (3 , 26) . In cross section, a narrow C-shaped region outlines the muscle, which is the orbital region that contains an orbital SIF (type 1) with a coarse appearance with the trichrome stain and an orbital MIF (type 2) with a fine, granular appearance. The larger global region contains three SIFs (types 3–5) and a global MIF (type 6). Each has a distinct trichrome staining pattern (3) . Histological images were obtained using a SPOT digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) and analyzed by Image-Pro plus software (Media Cybernetics, Silver Spring, MD). The degree of sarcolemmal staining was graded as intense (++), moderate (+), or absent (-).

Contractile studies
A Grass stimulator (Model S8800) was used to stimulate the muscles. The stimulator was connected to a stimulus isolation unit (Model SIU5, Grass Instrument Division) to minimize artifacts. The stimulus isolation unit was connected to a stimulus booster (Stimu-Splitter II, Med-Lab Instruments), which in turn was connected to the stimulation electrodes. The muscle length was adjusted so that the twitch tension was maximal. The length of the muscle (L0) was maintained throughout the experiment. Four or five animals were used for each drug. For baseline and drug treatments, the following stimulation protocol was used. The voltage and pulse duration were adjusted to obtain the maximal response from the muscle. Six twitches and six trains were recorded from each muscle during each experimental condition. Consecutive twitches at regular intervals of 30 s were first recorded. A fused tetanic force response always occurred at ~200 Hz for the lateral rectus. For tetanic contraction studies, 200 Hz stimulation was performed with a train duration of 333 ms and a train interval of 30 s.

Muscle stimulation records were digitized using the program Axotape (Axon Industries, Forest City, CA). The length of the muscle was measured. The muscle was blotted and weighed after removing the bone and tendon. Muscle area was calculated as muscle weight (grams) divided by the product of fiber length (centimeters) times the density of the muscle (1.06/cm (3) (27) . Because of the size variability of the muscles, the force was normalized and force generated (in grams) was converted to Newtons.

Solutions and drugs
L-NAME and spermineNONOate were purchased from Molecular Probes (Eugene, OR). S-nitrosoacetylcysteine (SNAC) was prepared as described by Kröncke and Kolb-Bachofen (28) . Cysteine HCl and NaNO2, used for production of SNAC, were purchased from Sigma Chemical (St. Louis, MO). ‘Spent’ SNAC was prepared by leaving the SNAC solution overnight at room temperature to react completely. The drugs were added directly to the physiological saline solution in the tissue–organ bath. The drugs were prepared fresh for each experiment and added to the tissue–organ bath to achieve the desired concentrations: L-NAME, 200 µM; SNAC, 1 mM; spermineNONOate, 2 mM. Baseline recordings were made before the addition of the drug. Fifteen minutes after addition of L-NAME solution, the stimulation protocol was repeated. In experiments with SNAC, spent SNAC was added after baseline recordings. Twitches and trains were repeated and recorded after 45 min. Then the tissue–organ bath was flushed and fresh physiological saline was added. Freshly prepared SNAC was added and the stimulation protocol was repeated after 45 min. SpermineNONOate spontaneously releases NO with a half-life of ~40 min (29 , 30) . Twitches and trains were recorded at 2 and 40 min after the addition of spermineNONOate. After 50 min, the tissue–organ bath was flushed and filled with fresh 37°C physiological saline solution. Stimulations were repeated after 40 min. Twitches and trains were recorded at 15, 30, and 60 min for studying the recovery of the contractile force after flushing the test solution. There was a gradual increase in the tension for up to 40 min and a decrease at 60 min.

Statistical analysis
The data were analyzed and tested for statistical significance (P<0.05) using ANOVA and one-sample t tests. Tables show mean values, the standard error, and the number of muscles studied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NADPH diaphorase histochemistry
As determined by NADPH diaphorase staining, nNOS expression in EOM was confined primarily to the sarcolemma (Fig. 1 ). Certain fibers demonstrated internal diaphorase stain. Such staining in other skeletal muscle is attributed to NADPH-reducing enzymes within mitochondria, but neuronal nNOS may also be expressed in mitochondria. In some areas, the NADPH diaphorase activity was concentrated at the edges of fibers with the morphology of an endplate, as described previously (24) . The fibers with stained sarcolemma followed a distinct anatomic distribution (Table 1) . The orbital and global MIFs, types 2 and 6, had no NADPH diaphorase stain, whereas the SIFs of all regions had intense NADPH diaphorase stained sarcolemma. Only the type 5 global SIF had qualitatively less NADPH diaphorase staining of the sarcolemma. No internal NADPH diaphorase staining was seen in the MIFs consistent with their low levels of mitochondria.



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  		Figure 1. Trichrome stain (A) and NADPH diaphorase stain (B) of serial sections of the global region of EOM. The majority of fibers have circumferential NADPH diaphorase staining of the sarcolemma. Asterisks mark the position of 2 global MIFs without NADPH diaphorase stain. The global MIFs are identified by the fine staining pattern with the Trichrome. All fibers with the course granular appearance of Trichrome have sarcolemmal staining with NADPH diaphorase stain and are type 3 fibers (see Table 1 for summary). Scale bar = 50 µm.

nNOS catalytic activity
Results of nNOS catalytic activity are summarized in Table 2 . EOM had the highest level of nNOS activity. EDL activity was significantly greater than diaphragm and soleus muscles (P<0.0001), but no difference existed between diaphragm and soleus (P<0.67).


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  		Table 2. nNOS activity in extracts from wild-type muscle

NO studies of contractility
Figure 2 shows the digitized records of 200 Hz tetanic contractions of the lateral rectus before and after the addition of the NO modulators. Table 3 shows the effect of NO-modulating agents on EOM twitch and tetanic tension. The nNOS inhibitor L-NAME increased the peak twitch tension by 28%. Peak tetanic tension showed a mean increase of 32% 15 min after addition of L-NAME.



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  		Figure 2. Original records of tetanic contractions. A) Before and 15 min after addition of L-NAME. B) Before and 40 min after addition of spermine-NONOate.


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  		Table 3. Effect of NO drugs on the twitch and tetanic tension of lateral rectus muscle at 37°C

The NO donors SNAC and spermineNONOate both significantly reduced the peak twitch and tetanic tensions. S-Nitrocysteine decomposes generating NO and cystine (21 , 28) . The effect on contractility of a solution that had been fully reacted (spent SNAC) was tested to assure that cystine did not contribute to alterations in contractility. Spent SNAC decreased the peak twitch tension by 6% and the peak tetanic tension by 12%. This reduction in the contractile force may be due to the action of cystine or the SNAC may not have been fully reacted or spent. SNAC produced a decrease of 33% in twitch and 22% in tetanic contractile force compared with the baseline values. The percentage reduction in force after subtracting the reduction in force due to the action of spent SNAC was 27% and 10%, respectively, for twitch and tetanus. This reduction in force noticed after the action of spent SNAC was interpreted as due to the action of NO.

SpermineNONOate produced a dramatic and rapid reduction in twitch tension. A mean decrease of 37% was observed after 2 min. The tension continued to decrease; a mean reduction of 59% occurred 40 min after addition of the drug. Mean peak tetanic contractions showed a 32% decrease after 2 min and a 38% decrease after 40 min. Flushing and addition of physiological saline resulted in an increase in contractile force, but not to predrug treatment levels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first characterization of nNOS expression and NO effects on EOM contraction. Neuronal NOS enzymatic activity was higher in EOM than with EDL, which had previously been identified as having the highest nNOS activity among skeletal muscles (11) . The results are consistent with the observation that nNOS activity correlates positively with the percentage of fast-twitch fibers and negatively with the level of force generation of a muscle (11) . Although nNOS activity is significantly greater statistically in EOM vs. the other muscles, the percentage difference in enzyme activity in comparison with EDL is small. However, because of the small size of EOM fibers, the influence on contraction of the short-diffusing NO may be magnified compared with other skeletal muscles.

NADPH diaphorase staining was localized to the sarcolemma of all the orbital and global SIF EOM fiber types. Although the fiber classification used in other skeletal muscle cannot be applied to the EOM, the EOM SIFs are most similar to the fast-twitch fibers of other skeletal muscle based on myosin isoform expression and oxidative capacity (26) . A true slow-twitch fiber is not observed in the primary EOMs. The present results are consistent with nNOS activity and expression in other skeletal muscles being limited to fast-twitch fibers (11 , 21) . The type 5 global SIF had qualitatively less NADPH diaphorase staining. The significance of this observation cannot be determined from this examination. This fiber type has low mitochondrial content, a fast ATPase profile, and modest levels of oxidative enzymes, and therefore has a low fatigue resistance. Because of this profile, the fiber type is thought to be recruited only sporadically during eye movements (26) . From a functional standpoint, nNOS expression may be less important for this type of fiber. The orbital and global MIF fibers had no NADPH diaphorase activity. The global MIFs contract in a graded or tonic fashion whereas the orbital MIFs have contractile characteristics intermediate to those of twitch and tonic fibers (9) . The degree of force generation in tonic fibers is dependent on the rate of neuronal stimulation. Therefore, NO generation is not expected to be a mechanism for modulation of contractile force in MIFs.

The NO donors SNAC and spermineNONOate decreased tetanic tension and maximum twitch, and the nNOS inhibitor L-NAME increased force generation of isolated lateral rectus. Skeletal muscle tonically produces NO, which functions as an endogenous modulator of muscle function at both the tissue and cellular levels (31) . Using the rat diaphragm, extensor digitorum longus, and soleus, Kobzik et al. (11) found during submaximal tetanic contractions a mean reduction of ~1% in isometric force for the NO donor SNAC and ~3% for another NO donor, sodium nitroprusside, whereas nNOS blockade with nitro-L-arginine at a 1 mM concentration increased contraction by ~8%. Under the experimental conditions used in this study, the effect of NO-modulating agents was much more dramatic on EOM than observed in other skeletal muscles. L-NAME increased force of the twitches by 28%; SNAC reduced isometric twitch tension by 26% and spermineNONOate produced a reduction of 60%. The more prominent reduction by spermineNONOate may be due to its rapid generation of NO and the effect of spermine (32) . Flushing and the addition of fresh physiological saline resulted in a 10% reversal in the contractile force of twitch tension and 18% reversal in the tetanic force. This partial reversal may be due to toxic effects of NO (33) or slowly reversible nitrotyrosine modification of proteins (34 , 35) .

The effect of NO on muscle contractility appears to be influenced by experimental design and the muscle studied. The force of contraction of soleus, a predominantly slow-twitch muscle, was increased by the NO donor nitroprusside (36) . The same group subsequently reported (37 , 38) that NO decreased the contractility of the canine soleus muscle in vitro and the gastrocnemius-plantaris of mouse in situ. They hypothesized that the differences in stimulation parameters contributed to the opposite conclusions reached by their studies. In the present study, the choice of NO donor significantly influenced the effect on NO contractility. Others have observed variations in response dependent on the choice of the NO-modulating agent (11 , 39) .

Peak twitch and tetanic contractile force of EOM are less than with other skeletal muscles even when normalized to the cross-sectional area (4 , 15 , 40 , 41) . Loss of fiber excitability, a greater amount of connective tissue, or low force output of the myofibrils have been postulated to explain the low levels of force generation of EOMs. In addition, different myofibrils may have fewer numbers of active cross bridges. However, single, skinned rabbit EOM fibers generate maximum isometric force levels similar to limb fibers, even though the normalized force response of the intact muscles is low (4 , 16) . Therefore, actomyosin cross bridge formation appears to be similar to limb muscles. The MIFs may not generate similar amounts of force as the SIFs and effectively reduce the total contractile force of EOM (4) . From the present study, the high level of NO generation coupled with the small fiber size of EOM may also serve to reduce force generation.

The mechanism of NO modulation of force generation in non-EOM skeletal muscle appears to operate via several mechanisms, but primarily by the modulation of cGMP pathways and myofibrillar adenosine triphosphatase (ATPase) activity. Inhibition of guanylyl cyclase increases force generation of the diaphragm, whereas cGMP analogs and phosphodiesterase inhibition reduce force (11) . EOM contractility is affected in a similar fashion (unpublished observations), but agents that modulate NO directly affect contraction to a greater degree than cGMP modulators. NO donors also decrease myofibrillar ATPase (42) and creatine kinase activity (43 , 44) , which, if occurring in EOM, would reduce contractility. Meszaros and Bak (45) showed that NO decreases the rate of Ca2+ release from intracellular stores (like sarcoplasmic reticulum), which may lead to diminished contractility. Galler and co-workers (42) observed that S-nitroso-N-acetylpenicillamine depressed both mechanical properties and myofibrillar ATPase activity of rat skeletal muscle. They suggested that NO has a direct inhibitory effect on the force-generating proteins in skeletal muscle. In studies of single fibers from the mouse, Andrade et al. (46) found that NO impairs Ca2+ activation of the actin filaments, which results in decreased myofibrillar Ca2+ sensitivity.

The present results suggest that NO plays a greater role in contraction of EOM than in other skeletal muscles. NO has a short diffusion range and interacts rapidly with its targets (47) . The small caliber of EOM fibers coupled with high rates of NO synthesis could contribute to NO affecting EOM contraction to a greater extent in vivo. During muscle contraction NO is generated by contracting muscle, which may hasten recovery from activity by dampening muscle contractile force, increasing glucose uptake and muscle blood flow (47 , 48) . EOMs have a rich vascular bed and are constantly active. In addition to effects on contraction, NO generated by contracting EOMs may be important in matching contractile activity with the metabolic requirements of the muscle.

In summary, nNOS is exclusively localized to the SIFs of EOM, which has high total levels of nNOS catalytic activity. In contrast to other skeletal muscles, NO-acting agents have greater effects on EOM contractility. The more prominent reduction of EOM contractile force by NO offers an additional mechanism to explain why EOM generates a low level of force compared with other skeletal muscles. This property of EOM likely is important in their specialized role in generation of eye movements and further adds to the complexity of this specialized muscle group.


   ACKNOWLEDGMENTS
 
Supported by the Office of Research and Development, Medical Research Service of the Department of Veterans Affairs, NIH grants EY-11998, EY-13238, and Vision Core grant EY-113373–01A1.

Received for publication December 21, 2000. Revision received April 16, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Porter, J. D., Baker, R. S. (1996) Muscles of a different ‘color’: the unusual properties of the extraocular muscles may predispose or protect them in neurogenic and myogenic disease. Neurology 46,30-37[Free Full Text]
  2. Ruff, R. L., Kaminski, H. J., Maas, E., Spiegel, P. (1989) Ocular muscles: physiology and structure–function correlations. Bull. Soc. Belg. Ophthalmol. 237,321-352
  3. Spencer, R. F., Porter, J. D. (1988) Structural organization of the extraocular muscles. Buttner-Ennever, J. eds. Neuroanatomy of the Oculomotor System ,33-79 Elsevier Amsterdam.
  4. Lynch, G. S., Frueh, B. R., Williams, D. A. (1994) Contractile properties of single skinned fibres from the extraocular muscles, the levator and superior rectus, of the rabbit. J. Physiol. 475,337-346[Abstract/Free Full Text]
  5. Demer, J. L., Oh, S. Y., Poukens, V. (2000) Evidence for active control of rectus extraocular muscle pulleys. Invest. Ophthalmol. Vis. Sci. 41,1280-1290[Abstract/Free Full Text]
  6. Shall, M. S., Goldberg, S. J. (1992) Extraocular motor units: type classification and motoneuron stimulation frequency-muscle unit force stimulation relationships. Brain Res 587,291-300[Medline]
  7. Sartore, S., Mascarello, F., Rowlerson, A., Gorza, L., Ausoni, S., Vianello, M., Schiaffino, S. (1987) Fibre types in extraocular muscles: a new myosin isoform in the fast fibres. J. Muscle Res. Cell Motil. 8,161-172[Medline]
  8. Wieczorek, D. F., Periasamy, M., Butler-Browse, G. S., Whalen, R. G., Nadal-Ginard, B. (1985) Co-expression of multiple myosin heavy chain genes in addition to a tissue-specific one in extraocular musculature. J. Cell Biol. 101,618-629[Abstract/Free Full Text]
  9. Jacoby, J., Chiarandini, D. J., Stefani, E. (1989) Electrical properties and innervation of fibers in the orbital layer of rat extraocular muscles. J. Neurophysiol. 61,116-125[Abstract/Free Full Text]
  10. Silvagno, F., Xia, H., Bredt, D. S. (1996) Neuronal nitric-oxide synthase-µ an alternatively spliced isoform expressed in differentiated skeletal muscle. J. Biol. Chem. 271,11204-11208[Abstract/Free Full Text]
  11. Kobzik, L., Reid, M. B., Bredt, D. S., Stamler, J. S. (1994) Nitric oxide in skeletal muscle. Nature (London) 372,546-548[Medline]
  12. Kobzik, L., Stringer, B., Balligand, J. L., Reid, M. B., Stamler, J. S. (1995) Endothelial-type nitric oxide synthase (ec-NOS) in skeletal muscle fibers: mitochondrial relationships. Biochem. Biophys. Res. Commun. 211,375-381[Medline]
  13. Kaminski, H. J., and Andrade, F. H. (2001) Nitric oxide: biologic effects on muscle and role in muscle disease. Neuromusc. Disord. In press
  14. Stamler, J. S., Meissner, G. (2001) Physiology of nitric oxide in skeletal muscle. Physiol. Rev. 81,209-237[Abstract/Free Full Text]
  15. Close, R., Luff, A. (1974) Dynamic properties of inferior rectus muscle of the rat. J. Physiol. 236,259-270[Medline]
  16. Frueh, B. R., Hayes, A., Lynch, G. S., Williams, D. A. (1994) Contractile properties and temperature sensitivity of the extraocular muscles, the levator and superior rectus, of the rabbit. J. Physiol. 475,327-336[Abstract/Free Full Text]
  17. Hanson, J., Lennerstrand, G. (1977) Contractile and histochemical properties of the inferior oblique muscle in the rat and the cat. Acta Ophthalmol 55,88-102
  18. Burke, R. (1981) Motor units: anatomy, physiology, and functional organization. Brooks, V. eds. Handbook of Physiology, Section 1: The Nervous System ,345-422 American Physiological Society Bethesda, MD.
  19. Park, J. H., Straub, V. A., O’Shea, M. (1998) Anterograde signaling by nitric oxide: characterization and in vitro reconstitution of an identified nitrergic synapse. J. Neurosci. 18,5463-5476[Abstract/Free Full Text]
  20. Brenman, J., Chao, D., Xia, H., Aldape, K., Bredt, D. (1995) Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 82,743-752[Medline]
  21. Richmonds, C. R., Boonyapisit, K., Kusner, L. L., Kaminski, H. J. (1999) Nitric oxide synthase in aging skeletal muscle. Mech. Ageing Dev. 109,177-189[Medline]
  22. Hope, B. T., Michael, G. J., Knigge, K. M., Vincent, S. R. (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc. Natl. Acad. Sci. USA 88,2811-2814[Abstract/Free Full Text]
  23. Dawson, T. M., Bredt, D. S., Fotuhi, M., Hwang, P. M., Snyder, S. H. (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc. Natl. Acad. Sci. USA 88,7797-7801[Abstract/Free Full Text]
  24. Kusner, L. L., Kaminski, H. J. (1996) Nitric oxide synthase is concentrated at the skeletal muscle endplate. Brain Res 730,238-242[Medline]
  25. Loughlin, M. (1993) Muscle Biopsy: A Laboratory Investigation Butterworth-Heinemann Oxford.
  26. Porter, J. D., Baker, R. S., Ragusa, R. J., Brueckner, J. K. (1995) Extraocular muscles: basic and clinical aspects of structure and function. Surv. Ophthalmol. 39,451-484[Medline]
  27. Mendez, J., Keys, A. (1960) Density and composition of mammalian muscle. Metabolism 9,184-188
  28. Kröncke, K.-D., Kolb-Bachofen, V. (1996) Detection of nitric oxide interaction with zinc finger proteins. Packer, L. eds. Nitric Oxide, Part B: Physiological and Pathological Processes 269,279-284 Academic Press San Diego.
  29. Redford, E., Kapoor, R., Smith, K. (1997) Nitric oxide donors reversibly block axonal conduction: demyelinated axons are especially susceptible. Brain 120,2149-2157[Abstract/Free Full Text]
  30. Christen, S., Gee, P., Ames, B. N. (1996) Mutagenicity of nitric oxide in base pair-specific Salmonella tester strains: TA7000 series. Packer, L. eds. Methods in Enzymology 269,267-278 Academic Press San Diego. [Medline]
  31. Reid, M. B. (1998) Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance. Acta Physiol. Scand. 162,401-409[Medline]
  32. De Meis, L. (1967) Relaxing effect of spermine and spermidine on intact and glycerol-treated muscle. Am. J. Physiol. 212,92-96
  33. Richmonds, C. R., Kaminski, H. J. (2000) Nitric oxide myotoxicity is age related. Mech. Ageing Dev. 113,183-191[Medline]
  34. Stamler, J. S. (1994) Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78,931-936[Medline]
  35. Viner, R. I., Williams, T. D., Schöneich, C. (1999) Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and s-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase. Biochemistry 38,12408-12415[Medline]
  36. Murrant, C. L., Woodley, N. E., Barclay, J. K. (1994) Effect of nitroprusside and endothelium-derived products on slow-twitch skeletal muscle function in vitro. Can. J. Physiol. Pharmacol. 72,1089-1093[Medline]
  37. Murrant, C. L., Frisbee, J. C., Barclay, J. K. (1997) The effect of nitric oxide and endothelin on skeletal muscle contractility changes when stimulation is altered. Can. J. Physiol. Pharmacol. 75,414-422[Medline]
  38. Murrant, C., Barclay, J. (1995) Endothelial cell products alter mammalian skeletal muscle function in vitro. Can. J. Physiol. Pharmacol. 73,736-741[Medline]
  39. Reid, M. B., Kobzik, L., Bredt, D. S., Stamler, J. S. (1998) Nitric oxide modulates excitation-contraction coupling in diaphragm. Comp. Biochem. Physiol. 119A,211-218
  40. Asmussen, G., Gaunitz, U. (1981) Mechanical properties of the isolated inferior oblique muscle of the rabbit. Pfluegers Arch 392,183-190[Medline]
  41. Andrade, F. H., Porter, J. D., Kaminski, H. J. (2000) Eye muscle sparing by the muscular dystrophies: lessons to be learned?. Microsc. Res. Tech. 48,192-203[Medline]
  42. Galler, S., Hilber, K., Göbesberger, A. (1997) Effects of nitric oxide on force-generating proteins of skeletal muscle. Pfluegers Arch 434,242-245[Medline]
  43. Wolosker, H., Panizzutti, R., Engelender, S. (1995) Inhibition of creatine kinase by S-nitrosoglutathione. FEBS Lett 392,274-276
  44. Gross, W. L., Bak, M. I., Ingwall, J. S., Arstall, M. A., Smith, T. W., Balligand, J.-L., Kelly, R. A. (1996) Nitric oxide inhibits creatine kinase and regulateless rat heart contractile reserve. Proc. Natl. Acad. Sci. USA 93,5604-5609[Abstract/Free Full Text]
  45. Meszaros, L. G., Bak, J. (1991) Simultaneous Ca binding to and occlusion within the sarcoplasmic reticulum Ca-pump which contradicts the E1-E2 model. Biophys. J. 59,252a(abstr.)
  46. Andrade, F. H., Reid, M. B., Allen, D. G., Westerblad, H. (1998) Effect of nitric oxide on single skeletal muscle fibres from the mouse. J. Physiol. 509,577-586[Abstract/Free Full Text]
  47. Beckman, J. S., Koppenol, W. H. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am. J. Physiol. 271,C1424-C1437[Abstract/Free Full Text]
  48. Espey, M. G., Miranda, K. M., Feelisch, M., Fukuto, J., Grisham, M. B., Vitek, M. P., Wink, D. A. (2000) Mechanisms of cell death governed by the balance between nitrosative and oxidative stress. Ann. N.Y. Acad. Sci. 899,209-221[Medline]



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