Heme oxygenase-2 is present in the sarcolemma region of skeletal muscle fibers and is non-continuously co-localized with nitric oxide synthase-1

There is increasing evidence that the heme oxygenase-2 (HO-2)/carbon monoxide (CO) pathway and the nitric oxide synthase (NOS)/nitric oxide (NO) pathway functionally cross-talk. Therefore, we investigated the appearance of HO-2 in mammalian skeletal muscles where NOS-1 is known to be expressed in high quantities. Immunoblotting of rat hind limb extensor muscles extracts revealed a single 36 kDa band demonstrating the existence of HO-2 in skeletal muscle and indicating the monospecifity of the antibody that was applied. Immunohistochemistry on healthy rat extensor hind limb muscles showed that HO-2 is present in satellite cells, endothelial cells of the vascular system, fibrocytes/fibroblasts but also fiber type-independently in extrafusal myofibers either in association with the non-junctional sarcolemma region, or in a subsarcolemmal network or, less prominently, in cross-striated stripes connected to longitudinally running lines. Combined HO-2 immunohistochemistry and NOS-1 histochemistry revealed an apparent co-localization of both molecules only in the non-junctional sarcolemma region of extrafusal type II myofibers outside costameres. In diseased muscles of mdx mice, HO-2 expression was not changed. In patients suffering from Duchenne's muscular dystrophy, it was absent in the sarcolemma region. In conclusion, the HO-2/CO system is present in mammalian skeletal muscle where it is non-continuously co-localized with the NOS-1/NO-system. This finding implicates an optionally functional cross-talk between both gaseous signaling pathways.     

The specificity of the histochemical NADPH diaphorase reaction for nitric oxide synthase-1 in skeletal muscles is increased in the presence of urea

Nitric oxide synthase-1 (NOS-1) can be demonstrated in the sarcolemma region of myofibers in rodent skeletal muscles with the use of NADPH diaphorase histochemistry. Since other, especially intrafibrar enzymes also exhibit NADPH diaphorase activity, we tried to increase the specificity of the histochemical reaction for NOS-1. A qualitative and quantitative analysis was performed on cryostat sections of fast-twitch oxidative myofiber-rich tongue and fast-twitch glycolytic myofibers-rich tibialis anterior muscle derived from C57 mice and NOS-1 deficient knockout mice. All myofibers of both C57 mice and NOS-1 knockout mice contained significant intrafibrar NADPH diaphorase activity which was inhibited to almost background levels when 2 M urea was added to the incubation medium. On the other hand, myofibers of C57 mice but not of NOS-1-deficient knockout mice exhibited NADPH diaphorase activity in their sarcolemma region which was only weakly reduced in the presence of 2 M urea as was demonstrated by image analysis. Quantitative data on the activity of NADPH diaphorase(s) were obtained in situ by photometric analysis of formazan extracted from cryostat sections. The catalytic activity in tongue and tibialis anterior muscle was reduced in presence of 2 M urea to approximately 27% in C57 mice and to 7-17% in NOS-1 knockout mice, respectively. An in vitro NADPH diaphorase assay performed on homogenates of skeletal muscles also revealed an inhibitory effect of 2 M urea in both mouse strains and, additionally, indicated an upregulation of NADPH diaphorase activity in NOS-1 knockout mice. Finally, an immunodepletion analysis demonstrated that NOS-1 comprises 38% of the total NADPH diaphorase activity in tongue and approximately 59% in tibialis anterior muscle in C57 mice. In conclusion, we recommend the addition of 2 M urea to the incubation medium to increase the specificity of the NADPH diaphorase reaction to localise NOS-1 with the use of catalytic histochemistry.     

Association of soluble guanylate cyclase with the sarcolemma of mammalian skeletal muscle fibers

Previous investigations have shown that NO-producing nitric oxide synthase (NOS)-1 and CO-generating heme oxygenase (HO-2) are associated with the sarcolemma of skeletal muscle fibers in many mammalian species. Despite numerous roles ascribed to NO and possibly also CO in skeletal muscle, a specific receptor for both gases has hitherto not been found in myofibers. Therefore, in the present work the appearance of the alpha1, beta1 and beta2 subunits of soluble guanylate cyclase (sGC), the most commonly known receptor for NO and potentially also CO, was analysed in mammalian skeletal muscles using immunoblotting and immunohistochemistry. Immunoblotting with an antibody against the beta1 subunit of sGC revealed a band of 70 kDa corresponding to the molecular weight of this protein. Immunohistochemistry with antibodies against the alpha1, beta1 and beta2 sGC subunits showed that the larger part of positivity was present in the sarcolemma region of skeletal muscle fibers and colocalized with NOS-1 mainly in type II myofibers and with HO-2 in type I and type II myofibers. For the first time, sarcolemmal association of sGC and its colocalization with NOS-1 generating the sGC-activator NO and with HO-2 producing the potential sGC upregulator CO have been demonstrated in the present study. These results enable a better understanding of the role of NO and CO in myofibers and suggest a so far unknown molecular mechanism for the interaction of sGC with the sarcolemma.     

Effects of perchlorate on myofibrillar calcium sensitivity in rat skinned skeletal muscles

The effects of perchlorate (1–20 mm ) on myofibrillar calcium responsiveness have been tested in Triton X-100-skinned fibre bundles from rat soleus (slow-twitch) and extensor digitorum longus (fast-twitch) skeletal muscles. In extensor digitorum longus and soleus, perchlorate dose-dependently shifted the pCa (-log[Ca²⁺])/tension relationship towards lower free calcium concentration (sensitizing effect) and maximal tension was unchanged. The degree of sensitization was greater in extensor digitorum longus than in soleus bundles. Reversibility after exposure to 12 mm perchlorate was complete in soleus but not in extensor digitorum longus muscles. In fact, the ‘return’ pCa/tension relationship in extensor digitorum longus was shifted to higher free calcium concentration (desensitizing effect) compared with control. Perchlorate (12 mm ) also enhanced myofibrillar calcium responsiveness of frog semitendinosus skinned skeletal fibres. Assuming a passive distribution of perchlorate across the sarcolemma, this sensitizing effect is probably not involved in perchlorate-induced potentiation of contractile responses of intact muscles and thereby supports the specificity of perchlorate as an agonist of the excitation/calcium release sequence in skeletal muscle fibres.     

Constitutive coexpression of nitric oxide synthase-1 and soluble guanylyl cyclase in myoepithelial cells of mammary glands in mice

An impact of nitric oxide (NO) on lactation and milk secretion in mammary glands has previously been documented, but the underlying molecular mechanisms for this modulatory effect remain unclear. Therefore, we investigated the expression patterns of NO synthase (NOS)-1, NOS-3 and the NO receptor soluble guanylyl cyclase (sGC) in mammary glands of lactating and non-lactating female C57/Bl6 mice. RT-PCR demonstrated the existence of NOS-1-mRNA and NOS-3-mRNA in both lactating and resting mammary tissue. Immunoblots loaded with equal amounts of homogenate proteins from lactating and resting mammary tissues revealed comparable intensities of NOS-1 and sGC bands. Performing catalytic NADPH diaphorase histochemistry and immunohistochemistry, NOS-1 was only detected in myoepithelial cells (MEC), while sGC was localized in alveolar epithelial cells (lactocytes) and MEC in both lactating and non-lactating mammary glands. The non-modulated co-expression of both enzymes suggests that NOS-1 and sGC contribute to the constitutive regulation of tone in MEC.     

Topographic comparison of neuromuscular junctions in mouse slow and fast twitch muscles

The neuromuscular junctions of mammalian slow and fast twitch muscles are activated differently in vivo and show corresponding physiological differences in vitro, but the structural basis or consequences of these differences are relatively unexplored. Therefore, neuromuscular junctions of mouse fast (extensor digitorum longus) and slow (soleus) twitch muscles were compared by use of new scanning and light microscopy techniques. In both muscles, the endplate appeared as an elliptical area raised to a variable extent above the surrounding sarcolemma and containing the primary clefts. In most soleus endplates, this raised surface area was considerably higher and wider and about three times larger than in extensor digitorum longus. In addition, the primary cleft area was about two-fold greater in soleus than in extensor digitorum longus, even though cleft length was the same. The primary clefts formed either an elliptical shape along the outer margin of the endplate with inward-directed branches or a group of relatively rectilinear dendritic branches orthogonally oriented to one another. The latter type was most frequent in soleus and the elliptical type in extensor digitorum longus. Corresponding patterns of nerve terminal arborizations were seen by light microscopy. Although nerve terminal areas were the same in fast and slow muscles, in the former, numerous diverticulae significantly increased the length of the nerve terminal outline. The possible physiological significance of the different synaptic structure of slow and fast muscle is discussed.     

Skeletal muscle and heart antioxidant defences in response to sprint training

Although endurance training enhances the antioxidant defence of different tissues, information on the effect of sprint training is scanty. We examined the effect of sprint training on rat skeletal muscle and heart antioxidant defences. Male Wistar rats, 16–17 weeks old, were sprint trained on a treadmill for 6 weeks. Total glutathione levels and activities of glutathione peroxidase, glutathione reductase, glutathione S-transferase and superoxide dismutase in heart and various skeletal muscles were compared in trained and control sedentary animals. Lactate dehydrogenase and citrate synthase enzyme activities were measured in muscle to test the effects of training on glycolytic and oxidative metabolism. Sprint training significantly increased lactate dehydrogenase activity in predominantly fast glycolytic muscles and enhanced total glutathione contents of the superficial white quadriceps femoris, mixed gastrocnemius and fast-glycolytic extensor digitorum longus muscles. Oxidative metabolic capacity increased in plantaris muscle only. Compared with the control group, glutathione peroxidase activities in gastrocnemius, extensor digitorum longus muscles and heart also increased in sprint trained rats. Glutathione reductase activities increased significantly in the extensor digitorum longus muscle and heart. Glutathione S-transferase activity was also higher in the sprint trained extensor digitorum longus muscle. Sprint training did not influence glutathione levels or glutathione-related enzymes in the soleus muscle. Superoxide dismutase activity remained unchanged in skeletal muscle and heart. Sprint training selectively enhanced tissue antioxidant defences by increasing skeletal muscle glutathione content and upregulating glutathione redox cycle enzyme activities in fast and mixed fibre leg muscles and heart.     

Immunohistochemical localization of cardiodilatin in myoendocrine cells of the cardiac atria

Summary Region-specific antibodies against synthetic N-terminal fragments of cardiodilatin (CDD) were raised in rabbits and used for the immunohistochemical detection of this new peptide hormone in the myoendocrine cells within the cardiac atria of several species. The peroxidase-antiperoxidase (PAP) and fluorescein isothiocyanate (FITC) immunohistochemical methods gave identical results of cardiodilatin-immunoreactivity (CDD-IR) within the tissue. In addition to the porcine right atrial appendage, myoendocrine cells with CDD-IR were also detected in the left atrium of porcine heart, as well as in other species such as dog and cat. The exact localization of the immunoreactivity in specific secretory granules was mostly related to the Golgi-area which is located on both nuclear poles of auricular myoendocrine cells. The results confirm that cardiodilatin is stored in secretory granules observed through electron microscopical means. This hormone is most likely synthesized and released in myoendocrine cells, exerting its important cardiovascular effects.Supported by the Deutsche Forschungsgemeinschaft Carvas SFB 90     

Effect of diffusion distance on measurement of rat skeletal muscle glucose transport in vitro.

The relationships between muscle size, diffusion distance, and glucose uptake were studied using the Type IIb epitrochlearis (13 +/- 1 mg intact), Type I soleus (25 +/- 1 mg), and mixed Type IIa/IIb extensor digitorum longus (25 +/- 1 mg) from 60-70 g rats. Using intact muscles, the relative rates of 3-O-methyl-glucose uptake in response to 2 mUml-1 insulin were soleus = epitrochlearis greater than extensor digitorum longus, a finding inconsistent with the fibre-type compositions and the relative GLUT-4 protein levels (soleus greater than extensor digitorum longus greater than epitrochlearis). To test whether these results were influenced by substrate diffusion limitations in the tubular muscles, soleus and extensor digitorum longus were split longitudinally from tendon to tendon into strips of comparable size (13 +/- 1 mg) to the epitrochlearis. Insulin-stimulated rates of 3-O-methyl-glucose uptake were significantly enhanced in the split soleus (+120%) and split extensor digitorum longus (+200%), but not in the epitrochlearis, with the relative rates being soleus greater than extensor digitorum longus greater than epitrochlearis. Diffusion distances of the split soleus and extensor digitorum longus, as reflected by [14C]mannitol space equilibration time, were markedly enhanced (by at least 50%) relative to the intact muscles, and were comparable to that of the epitrochlearis. These results indicate that when muscles of different size and/or shape are used for in vitro measurement of glucose transport, the muscle preparations used must have similar diffusion distances for physiologically meaningful comparisons to be made.     

家兔趾长伸肌肌内神经、运动终板和肌梭的分布

目的：探究家兔趾长伸肌肌内神经、运动终板与肌梭的分布。方法：改良Sihler’s染色法、乙酰胆碱酯酶染色法及HE染色法。结果：趾长伸肌是短肌束构成的长肌,肌束起点高的,止点相应亦较高。连于第2趾的肌纤维占据了肌腹上1／3,止于其余3趾的肌纤维位于下2／3。趾长伸肌的神经来自2条肌外神经干,上支及其分支支配第2趾的肌纤维,下支司其余3趾。运动终板分布除冠状切面上为弥漫的黑色颗粒外,其余各切面均为一条连续的运动终板带。各部的肌梭密度分别是：起端33.95个／g、肌腹中部44.76个／g、止端为零。结论：家兔趾长伸肌内,肌梭分布不均匀。肌内神经分支密集的部位,运动终板集聚、肌梭密度亦高。家兔趾长伸肌具有划分亚部的特征。     

Morphological and biochemical correlations in muscle tissue of the atria and ventricles

An electron-microscopic investigation of heart muscle tissue showed that the cardiomyocytes of the atria differ from those of the ventricles. Three types of cells differing in the character of relations between the organoids and the number of specific secretory granules in them, were found in the atrial muscle tissue. A study of the distribution of the activity of lactate dehydrogenase (LD) and its isozymes by disc electrophoresis and isoelectric focusing showed that LD isozyme spectra of the ventricles and atria differ considerably from each other. It is suggested that the sources of energy and the mechanism of energy production are not the same in the muscle cells of the atria and ventricles.Department of Histology and Biological Chemistry, D. I. Ul'yanov Kuibyshev Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 484–486, April, 1978.     

Immunocytochemical evidence for CDD/ANP-like peptides in strands of myoendocrine cells associated with the ventricular conduction system of the rat heart

Summary Immunohistochemical investigations with different antisera against cardiodilatin 99–126 or alpha atrial natriuretic polypeptide revealed the presence of cardiac hormones not only in the atria of rats but also in strands of myoendocrine cells located in subendocardial regions of the ventricular septum. The localization of CDD-IR (cardiodilatin immunoreactivity) in the ventricle is associated with the location of the conduction system in the rat. The significance of the morphological relationship between cardiodilatin and the conduction system of the rat heart is discussed.     

Effect of diffusion distance on measurement of rat skeletal muscle glucose transport in vitro

The relationships between muscle size, diffusion distance, and glucose uptake were studied using the Type II b epitrochlearis (13 ± 1 mg intact), Type I soleus (25± 1 mg), and mixed Type II a/II b extensor digitorum longus (25 ± 1 mg) from 60–70 g rats. Using intact muscles, the relative rates of 3-O-methyl-glucose uptake in response to 2 mUml^-1 insulin were soleus = epitrochlearis > extensor digitorum longus, a finding inconsistent with the fibre-type compositions and the relative GLUT-4 protein levels (soleus > extensor digitorum longus > epitrochlearis). To test whether these results were influenced by substrate diffusion limitations in the tubular muscles, soleus and extensor digitorum longus were split longitudinally from tendon to tendon into strips of comparable size (13 ± 1 mg) to the epitrochlearis. Insulin-stimulated rates of 3-O-methyl-glucose uptake were significantly enhanced in the split soleus (+120%) and split extensor digitorum longus (+200%), but not in the epitrochlearis, with the relative rates being soleus > extensor digitorum longus > epitrochlearis. Diffusion distances of the split soleus and extensor digitorum longus, as reflected by [¹⁴C]mannitol space equilibration time, were markedly enhanced (by at least 50%) relative to the intact muscles, and were comparable to that of the epitrochlearis. These results indicate that when muscles of different size and/or shape are used for in vitro measurement of glucose transport, the muscle preparations used must have similar diffusion distances for physiologically meaningful comparisons to be made.     

Relative capabilities of sarcoplasmic reticulum in fast and slow mammalian skeletal muscles.

1. The calcium uptake capabilities of the sarcoplasmic reticulum (SR) of the fat-twitch muscles extensor digitorum longus (EDL) and tibialis anterior (TA) of the rat and the extensor digitorum longus of the cat have been compared with the same capabilities of the slow-twitch soleus muscles of the rat and cat. 2. For the ra the Vmax values of sarcoplasmic reticulum from tibialis anterior, extensor digitorum longus and from soleus muscles were 50, 51, and 10 micronmole Ca2+/g per minute, respectively. 3. For the extensor digitorum longus and soleus muscles of the cat the Vmax values were 34 and 5-6 micronmole Ca2+/g per minute, respectively. 4. These data were compared with mechanical data as reported in the literature for the same muscles. The relative calcium uptake capabilities of sarcoplasmic reticulum from slow and fast muscles corresponded closely to the relative rates of relaxation of these muscles.     

Postnatal appearance of 5-HT2A receptors on fast flexor and slow extensor rat motor neurons

Motor neurons to the slowly contracting extensor soleus muscle in behaving rats begin to fire tonically in the 2nd week after birth. In the adult, tonic firing becomes predominant and appears to arise from plateau potentials under monoaminergic control. In the present work, motor neurons to slowly contracting extensor soleus and rapidly contracting extensor digitorum longus, a physiological flexor muscle, were retrogradely labeled with fluorescent dextran and examined for immunoreactivity to 5-HT(2A) receptors in 1 and 2 week old and adult rats. No reactivity was detected at 1 week. At 2 weeks, reactivity was detected on 67% slowly contracting extensor soleus (16 of 24) and 19% extensor digitorum longus (11 of 57) motor neurons. In the adult, the intensity of staining was higher and the percentage of labeled motor neurons 79 for slowly contracting extensor soleus (34 of 43) and 31 for extensor digitorum longus (11 of 35). On slowly contracting extensor soleus motor neurons, labeling appeared more often on soma and dendrites than on dendrites only, whereas on extensor digitorum longus motor neurons, labeling appeared more often on dendrites only. These results are consistent with the hypothesis that serotonergic innervation contributes to the appearance and subsequent increase in tonic firing of rat slowly contracting extensor soleus motor neurons in postnatal development.     

Anatomical study on the extensor digitorum profundus muscle in the Japanese

An anatomical study on the extensor digitorum profundus muscle was made using 832 upper limbs from 416 Japanese adults. The separate muscles derived from the extensor digitorum profundus consist of 10 kinds: namely, the extensor pollicis longus, extensor pollicis et indicis accessorius, extensor indicis radialis, extensor indicis proprius, extensor indicis ulnaris, extensor indicis et medii accessorius, extensor medii proprius, extensor annularis proprius, extensor carpi profundus and extensor digiti brevis. The configuration of the muscles (except for the extensor digiti brevis) in the upper limb was classified into 13 types according to their arrangement and insertion. The most frequent type involved coexistence of the extensor pollicis longus and the extensor indicis proprius: it was observed in 664 limbs (79.8%). The next type involved coexistence of the extensor pollicis longus, extensor indicis proprius and the extensor medii proprius: it was observed in 67 limbs (8.1%). It appears that the extensor digiti brevis of man is derived from the most ulnar part of the extensor digitorum profundus which does not migrate proximally.     

NADPH-diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface.     

Effects of nerve cross-union on rat intracellular potassium in fast-twitch and slow-twitch rat muscles

1. Electrolytes of normal, self-innervated and cross-innervated extensor digitorum longus and soleus muscles of rats have been determined.2. [K](i) was 173 m-equiv/l. for both normal and self-innervated extensor digitorum longus. In cross-innervated extensor digitorum longus it was reduced to 159 m-equiv/l.3. For normal and self-innervated soleus, [K](i) was 150 m-equiv/l. and 154 m-equiv/l. respectively. In cross-innervated soleus it was increased to 182 m-equiv/l.4. The content and distribution of most other electrolytes of cross-innervated soleus, as well as its weight, were not significantly different from those of controls. On the other hand, cross-innervated extensor digitorum longus weighed about half as much as controls and contained markedly elevated Na(+), Cl(-) and extrafibre water and reduced non-collagenous protein and intrafibre water.5. It is concluded that [K](i) of fast-twitch and slow-twitch muscle fibres are under neural regulation. Possible mechanisms for this regulation are discussed.