Simultaneous labelling of basal lamina components and acetylcholinesterase at the neuromuscular junction

Summary A double labelling technique has been developed which permits the concomitant localization of basal lamina constituents together with acetylcholinesterase in mouse skeletal muscles. First, using the protein A-gold technique, type IV collagen and laminin were revealed on basal laminae ensheathing skeletal muscle fibres. The immunolabelling for both proteins was higher in synaptic than extrasynaptic regions. At synaptic sites the anti-type IV collagen immunolabelling exhibited an asymmetry; it was more intense on the portion of basal lamina closest to the postsynaptic membrane, whereas the anti-laminin immunolabelling was more uniformly distributed. It was also observed that the laminin immunoreactivity associated with Schwann and perineural cells was higher than that of skeletal muscle fibres. Secondly, the two basal lamina antigens were revealed simultaneously with another synaptic protein, acetylcholinesterase, using a refined cytochemical technique prior to the immunolabelling. The cytochemical reaction, which facilitates the location of endplates, did not alter the immunolabelling pattern. This double labelling procedure permits ready comparison of the distributions of type IV collagen and laminin with that of acetylcholinesterase, and may prove to be a useful approach in studies on synaptic components in developing and diseased muscle.     

Immunohistochemical localization of taurine in the male reproductive organs of the rat.

The amino acid taurine has been implicated in several aspects of reproductive system physiology. However, its localization in these organs has not been previously analyzed. The aim of this study was to characterize its distribution in male rat reproductive organs by immunohistochemical methods. Taurine was localized in the smooth muscle cells of the tissues studied and in the skeletal fibers of the cremaster muscle. In the testis, taurine was found in Leydig cells, vascular endothelial cells, and other interstitial cells. No immunoreactivity was observed in the cells of the seminiferous tubules, either in germ cells at all spermatogenic stages or in Sertoli cells. However, peritubular myoid cells were immunostained. Most epithelial cells of the efferent ducts were immunolabeled, whereas the epithelial cells of the rete testis (extratesticular segments), epididymis (caput, corpus, and cauda regions), and ductus deferens were unstained. However, most epithelial cells from the intratesticular segments of the rete were immunopositive. Some cells identified as intraepithelial macrophages and lymphocytes, apical cells, and narrow cells were intensely immunolabeled. Regional differences in the distribution of these cell types along the ducts studied were also noted. The possible functional roles for taurine in these cells are discussed.     

Regulation of Taurine Transport in Rat Skeletal Muscle

Taurine concentration of soleus muscle (SL, slow-twitch) was initially about twofold higher than that of extensor digitorum longus muscle (EDL, fast-twitch). Taurine concentration in gastrocnemius muscle (GC) was intermediate between that of EDL and SL. Four days after sciatic nerve section, taurine concentration in the EDL but not in the SL was increased by 2.5-fold. The increase was not due to the muscle atrophy and was observed 28 days after denervation. Tenotomy did not increase the total taurine content of the EDL. The increase in taurine concentration of the denervated EDL was prevented by simultaneous ingestion of guanidinoethane sulfonate, a competitive inhibitor of taurine transport. The initial and the maximal rates of [3H]taurine uptake were significantly higher in SL than in EDL. Denervation dramatically accelerated the initial and the maximal rates of the transport in EDL, whereas it significantly reduced those in SL. In contrast, the electrical stimulation of sciatic nerve accelerated the uptake of taurine by EDL and SL of the control but not of the curare-treated rats. These results suggest that transport of taurine into rat skeletal muscles is regulated differently by neural information and by muscular activity, and that the regulation is dependent on the muscle phenotype.     

Substance P immunoreactivity in rat von Ebner's gland

Summary The present immunohistochemical study revealed substance P-immunoreactive neuronal elements in the von Ebner's gland of rats. Immunoreactive ganglion cells were observed as single cells or groups of several immunoreactive ganglion cells among intra-lingual muscles, at the base of the vallate papillae and near the von Ebner's gland. Very numerous substance P-immunoreactive varicose nerve fibres ran closely associated with the serous cells and excretory duct cells, and were seen to run along blood vessels in the gland. Since substance P-immunoreactive ganglion cells were present near the glands, the immunoreactive varicose nerve fibres in the von Ebner's gland may be partly derived from the intra-lingual ganglion cells. These substance P-immunoreactive varicose nerve fibres may have an effect on the secretory activity of the serous cells and duct cells, and on the vasodilation of blood vessels of the von Ebner's gland. Actin immunoreactivity was seen in numerous myoepithelial cells embracing serous cells and duct cells, and in the smooth muscle cells of blood vessels of the gland. By using a double immunolabelling technique with anti-substance P and anti-actin sera, substance P-immunoreactive varicose nerve fibres were found to be in close contact with myoepithelial cells.     

Cardiac and skeletal muscle abnormality in taurine transporter-knockout mice

Taurine, a sulfur-containing β-amino acid, is highly contained in heart and skeletal muscle. Taurine has a variety of biological actions, such as ion movement, calcium handling and cytoprotection in the cardiac and skeletal muscles. Meanwhile, taurine deficiency leads various pathologies, including dilated cardiomyopathy, in cat and fox. However, the essential role of taurine depletion on pathogenesis has not been fully clarified. To address the physiological role of taurine in mammalian tissues, taurine transporter-(TauT-) knockout models were recently generated. TauTKO mice exhibited loss of body weight, abnormal cardiac function and the reduced exercise capacity with tissue taurine depletion. In this chapter, we summarize pathological profile and histological feature of heart and skeletal muscle in TauTKO mice.     

Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free β-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the β-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor α1 (Cbfα1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfα1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.     

UPTAKE AND RELEASE OF TAURINE FROM CEREBRAL CORTEX SLICES AND THEIR SUBCELLULAR COMPARTMENTS

—Cortex slices, synaptosomes and C-6 glioma cells were used to study [³⁵S]taurine uptake and its electrically-stimulated release. After exposure to taurine at two concentrations, the synaptosome preparation subsequently derived from the slices contained 41% of the particle-bound taurine and 16% of the total in the tissue. The uptake of [¹⁴C]GABA by C-6 glioma cells was inhibited 3-fold more by β-alanine than by l -DABA, whilst synaptosome preparations showed the opposite pattern, l -DABA being 2 or 3 times more effective than β-alanine. [³⁵S]Taurine uptake inhibition by l -DABA was low for synaptosomes and C-6 glioma, whereas β-alanine showed considerable effect on C-6 glioma (41%) and slices of white matter (ependyma; 50%). Synaptosome preparations showed little effect with β-alanine. When 30 min rather than 5 min incubations were employed, β-alanine depressed [³⁵S]taurine uptake by cortex slices by 30%. Taurine was taken up by a calcium-dependent mechanism and subcellular fractionation indicated that the synaptosome fraction showed losses commensurate with the net taurine release when low stimulation currents were used.     

Taurine in the osmoregulation of the Brattleboro rat

The function of taurine in mammalian osmoregulation was studied in the Brattleboro rat with hereditary hypothalamic diabetes insipidus (DI). DI rats are chronically dehydrated because of their inability to synthesize vasopressin. One day of water deprivation did not affect the water balance in rats with normal vasopressin synthesis, whereas DI rats were markedly dehydrated and lost considerably body weight. Taurine content and 3H-taurine accumulation by platelets were significantly higher in DI rats, with a further increase after one day of water deprivation. In DI rats, water deprivation also evoked a clear taurine increase in skeletal muscle and in the brain. These findings indicate that taurine has an osmoregulatory function in mammals.     

Taurine supplementation decreases oxidative stress in skeletal muscle after eccentric exercise

Infrequent exercise, typically involving eccentric actions, has been shown to cause oxidative stress and to damage muscle tissue. High taurine levels are present in skeletal muscle and may play a role in cellular defences against free radical‐mediated damage. This study investigates the effects of taurine supplementation on oxidative stress biomarkers after eccentric exercise (EE). Twenty‐four male rats were divided into the following groups (n = 6): control; EE; EE plus taurine (EE + Taurine); EE plus saline (EE + Saline). Taurine was administered as a 1‐ml 300 mg kg^?1 per body weight (BW) day^?1 solution in water by gavage, for 15 consecutive days. Starting on the 14th day of supplementation, the animals were submitted to one 90‐min downhill run session and constant velocity of 1·0 km h^?1. Forty‐eight hours after the exercise session, the animals were killed and the quadriceps muscles were surgically removed. Production of superoxide anion, creatine kinase (CK) levels, lipoperoxidation, carbonylation, total thiol content and antioxidant enzyme were analysed. Taurine supplementation was found to decrease superoxide radical production, CK, lipoperoxidation and carbonylation levels and increased total thiol content in skeletal muscle, but it did not affect antioxidant enzyme activity after EE. The present study suggests that taurine affects skeletal muscle contraction by decreasing oxidative stress, in association with decreased superoxide radical production. Copyright © 2010 John Wiley & Sons, Ltd.     

Immunohistochemical study on the distribution of vasoactive intestinal polypeptide (VIP) containing nerve fibers in the chicken pancreas

The distribution of vasoactive intestinal polypeptide (VIP) containing nervous elements in the chicken pancreas was immunohistochemically investigated by light microscopy. Strongly VIP immunoreactive ganglia existed in the interlobular connective tissue. Ganglion containing both VIP immunoreactive and non-immunoreactive nerve cells was occasionally observed in the connective tissue. Almost all the ganglion cells also showed acetylcholinesterase (AChE) activity. No extrapancreatic nerve bundles containing VIP immunoreactive nerve fibres were detected. VIP immunoreactive nerve fibres formed plexuses in the subepithelial layer of secretory ducts and the muscle layer of small arteries. The distribution pattern of VIP immunoreactive nerve fibers was similar to that of AChE-positive nerve fibers on adjacent sections. The exocrine pancreas received a rich supply of varicose nerve fibers showing VIP immunoreactivity. B-islets also were richly innervated by VIP immunoreactive varicose nerve fibers, whereas A-islets, only poorly. These observations suggest that VIP containing nerves in the chicken pancreas have an intrinsic origin, are probably derived from VIP immunoreactive, intrapancreatic ganglion cells and innervate secretory ducts, arteries, acinar cells and B-islets, and that VIP must coexist with acetylcholine in the nervous elements.     

Taurine deficiency,synthesis and transport in the mdx mouse model for Duchenne Muscular Dystrophy

The amino acid taurine is essential for the function of skeletal muscle and administration is proposed as a treatment for Duchenne Muscular Dystrophy (DMD). Taurine homeostasis is dependent on multiple processes including absorption of taurine from food, endogenous synthesis from cysteine and reabsorption in the kidney. This study investigates the cause of reported taurine deficiency in the dystrophic mdx mouse model of DMD. Levels of metabolites (taurine, cysteine, cysteine sulfinate and hypotaurine) and proteins (taurine transporter [TauT], cysteine deoxygenase and cysteine sulfinate dehydrogenase) were quantified in juvenile control C57 and dystrophic mdx mice aged 18 days, 4 and 6 weeks. In C57 mice, taurine content was much higher in both liver and plasma at 18 days, and both cysteine and cysteine deoxygenase were increased. As taurine levels decreased in maturing C57 mice, there was increased transport (reabsorption) of taurine in the kidney and muscle. In mdx mice, taurine and cysteine levels were much lower in liver and plasma at 18 days, and in muscle cysteine was low at 18 days, whereas taurine was lower at 4: these changes were associated with perturbations in taurine transport in liver, kidney and muscle and altered metabolism in liver and kidney. These data suggest that the maintenance of adequate body taurine relies on sufficient dietary intake of taurine and cysteine availability and metabolism, as well as retention of taurine by the kidney. This research indicates dystrophin deficiency not only perturbs taurine metabolism in the muscle but also affects taurine metabolism in the liver and kidney, and supports targeting cysteine and taurine deficiency as a potential therapy for DMD.     

Impaired cell volume regulation in taurine deficient cultured astrocytes

Taurine concentration was reduced by 40 and 65%, respectively in rat cerebellar astrocytes grown in a chemically defined medium or in culture medium containing a blocker of taurine transport (GES). Cell volume in these taurine deficient cells was 10%–16% higher than in controls. When challenged by hyposmotic conditions, astrocytes release taurine and this efflux contributes to the volume regulatory decrease observed in these cells. Taurine deficient astrocytes showed a less efficient volume recovery as compared to controls with normal taurine levels. Exposed to 50% hyposmotic medium, astrocytes with normal taurine concentration recovered 60% of their original volume whereas taurine deficient cells recovered only 30–35%. Similarly, in 30% hyposmotic medium, taurine deficient astrocytes recovered only 40% as compared to 75% in controls. No compensatory increases in the efflux of other osmolytes (free amino acids or potassium) were observed during regulatory volume decrease in taurine deficient astrocytes.     

Immunocytochemical localization of 67 KD Ca2+ binding protein (p67) in ventricular, skeletal, and smooth muscle cells.

By using immunocytochemical techniques, we examined the localization of a 67 kDa Ca2+ binding protein (p67) and calpactin I heavy chain (p36) in ventricular myocytes, skeletal myocytes, and intestinal smooth muscle cells. Immunofluorescence microscopy revealed that the p67 was expressed in all these muscle cells, whereas anti-p36 antibody stained cells in connective tissues but failed to stain these muscle cells. Immunogold electron microscopy was carried out to examine the subcellular localization of the p67 in muscle cells. The results showed that the p67 was exclusively confined to the plasma membrane of muscle cells and the presumptive transverse tubules of the striated myocytes. Immunoblot analysis with anti-p67 antibody showed that the p67 was indeed a constitutive protein of the sarcolemma isolated from rat hearts. These results indicate that the p67 is a sarcolemma-associated Ca2+ binding protein expressed in both striated myocytes and intestinal smooth muscle cells.     

Taurine promotes connective tissue growth factor (CTGF) expression in osteoblasts through the ERK signal pathway

Summary. Taurine is found in bone tissue, but its function in skeletal tissue is not fully understood. The present study was undertaken to investigate regulation of gene expression of connective tissue growth factor (CTGF), and the roles of mitogen-activated protein kinases (MAPKs) in murine osteoblast MC3T3-E1 cells treated with taurine. Western blot analysis showed taurine stimulated CTGF protein secretion in a dose- and time-dependent manner. Taurine induced activation of extracellular signal-regulated kinase (ERK), but not p38 and c-jun N-terminal Kinase (JNK), in osteoblasts. Furthermore, pretreatment of osteoblasts with the ERK inhibitor PD98059 abolished the taurine-induced CTGF production. These data indicate that taurine induces CTGF secretion in MC3T3-E1 cells mediated by the ERK pathway, and suggest that osteoblasts are direct targets of taurine.     

Potential benefits of taurine in the prevention of skeletal muscle impairment induced by disuse in the hindlimb-unloaded rat

Hindlimb unloading (HU) in rats induces severe atrophy and a slow-to-fast phenotype transition in postural slow-twitch muscles, as occurs in human disuse conditions, such as spaceflight or bed rest. In rats, a reduction of soleus muscle weight and a decrease of cross-sectional area (CSA) were observed as signs of atrophy. An increased expression of the fast-isoform of myosin heavy chain (MHC) showed the phenotype transition. In parallel the resting cytosolic calcium concentration (restCa) was decreased and the resting chloride conductance (gCl), which regulates muscle excitability, was increased toward the values of the fast-twitch muscles. Here, we investigated the possible role of taurine, which is known to modulate calcium homeostasis and gCl, in the restoration of muscle impairment due to 14-days-HU. We found elevated taurine content and higher expression of the taurine transporter TauT in the soleus muscle as compared to the fast-twitch extensor digitorum longus (EDL) muscle of control rats. Taurine level was reduced in the HU soleus muscle, although, TauT expression was not modified. Taurine oral supplementation (5?g/kg) fully prevented this loss, and preserved resting gCl and restCa together with the slow MHC phenotype. Taurine supplementation did not prevent the HU-induced drop of muscle weight or fiber CSA, but it restored the expression of MURF-1, an atrophy-related gene, suggesting a possible early protective effect of taurine. In conclusion, taurine prevented the HU-induced phenotypic transition of soleus muscle and might attenuate the atrophic process. These findings argue for the beneficial use of taurine in the treatment of disuse-induced muscle dysfunction.     

Taurine distribution in rat tissues during development.

1. Taurine levels have been determined in eight rat organs. 2. During postnatal growth the taurine content in retina, heart, small intestine, spleen and lung increases with advancing age, although adult values are not reached at the same time. 3. In contrast the taurine content decreases with age in brain cortex, liver and kidney. 4. The taurine in subcellular fractions of adult, 20-day-old and 5-day-old rat tissues exists predominantly in the cytosol of the cell. Taurine content in particulate fractions shows marked variations during development in the different organs. 5. Taurine distribution in the subcellular fractions suggests that some of the cellular taurine in the tissues is not freely mobile in cytosol.     

Taurine and Its Chloramine: Modulators of Immunity

Taurine is a semiessential amino acid that is not incorporated into proteins. In mammalian tissues, taurine is ubiquitous and is the most abundant free amino acid in the heart, retina, skeletal muscle, and leukocytes. Taurine reaches up to 50 mM concentration in leukocytes. Taurine has been shown to be tissue-protective in many models of oxidant-induced injury. One possibility is that taurine reacts with HOCl, produced by the myeloperoxidase (MPO) pathway, to produce the more stable but less toxic taurine chloramine (Tau-Cl). However, data from several laboratories demonstrate that Tau-Cl is a powerful regulator of the immune system. Specifically, Tau-Cl has been shown to downregulate the production of proinflammatory mediators in both rodent and human leukocytes. Recent molecular studies on the function of taurine provide evidence that taurine is a constituent of biological macromolecules. Specifically, two novel taurine-containing modified uridines have been found in both human and bovine mitrochondria. In studies on mechanism of action, Tau-Cl inhibits the activation of NFkappaB, a potent signal transducer for inflammatory cytokines, by oxidation of IkappaB alpha at methionine45. Taurine transporter knockout mice show reduced taurine, reduced fertility, and loss of vision resulting from severe retinal degeneration, which was found to be due to apoptosis. Apoptosis induced by amino chloramines is a current and important finding because oxidants derived from leukocytes play a key role in killing pathogens. The fundamental importance of taurine in adaptive and acquired immunity will be revealed using genetic manipulation.     

Ultrastructure of arterioles in the cat brain

Summary A total of 110 arterioles were examined in the brains of cats; different sites were studied including the cortex, putamen, pons and crus cerebri. No internal elastic laminae were seen in the subendothelial space, although occasional fragments of elastic material were present in the larger arterioles. The media was composed of one, two or three layers of smooth muscle cells which interlocked in such a way that the vessel wall thickness was constant. Numerous tight junctions were seen between adjacent smooth muscle cells and between the endothelium and smooth muscle cells. Apart from the usual cell organelles, the smooth muscle cells of arterioles had numerous dense patches on the cell surface. The structure of the adventitia varied according to the diameter of the vessel and the site in the brain; it contained adventitial cells, bundles of collagen fibres and nerve fibres. Innervation of arterioles was more constant in the brain stem than in the cortex. Metarterioles had less specialised, atypical smooth muscle cells, a discontinuous media and numerous, extensive myoendothelial tight junctions; they were not innervated by nerve fibres. The diameter of metarterioles was less than 10 m whereas that of arterioles was 10–45 m. The possible functional aspects of arteriolar innervation are discussed.     

Leiomyosarcomas: three cases with desmin positive tumour cells, lacking ultrastructural features of smooth muscle cells

A combined study of light and electron microscopy and of immunolabelling of three pleomorphic spindle cell sarcomas is presented. The light and electron microscopic features of these sarcomas were most compatible with those described for malignant fibrous histiocytoma (MFH, pleomorphic-storiform subtype). Electronmicroscopically undifferentiated and fibroblast-like cells, fibrohistiocytes and multinucleated histiocytes were observed. Characteristics belonging to smooth muscle cells were absent. By immunostaining, vimentin and desmin could be observed in tumour cells of all three cases, at least on frozen sections. Other markers such as alpha 1-antichymotrypsin, S-100 proteins, laminin, collagen IV and markers specific for skeletal muscle cells (myoglobin, actin and myosin specific for skeletal muscle) could not be demonstrated. These findings indicate that three MFH's are, in fact, poorly differentiated variants of smooth muscle tumours. It is concluded that immunophenotyping is very useful for this type of neoplasm.     

Subcellular immunolocalisation of fatty acid translocase (FAT)/CD36 in human type-1 and type-2 skeletal muscle fibres

Limited information exists about the putative role and expression in human skeletal muscle cells of the 88-kDa integral membrane protein fatty acid translocase (FAT), highly homologous to the human leucocyte differentiation factor CD36. Therefore, we investigated in healthy male individuals the muscle (m. vastus lateralis) fibre type specific expression and subcellular localisation of FAT/CD36. For this purpose four different monoclonal antibodies raised against human and mouse FAT/CD36 were used. Acetone or methanol/acetone fixation were tested. Serial cryosections were cut at –20°C, thaw-mounted on uncoated glass slides and air-dried before processing indirect immunofluorescence assays. Images were examined in a Nikon ER800 microscope, digitally captured, processed and analysed by LUCIA laboratory software. Three antibodies showed that FAT/CD36 was: (1) most abundantly expressed in capillary endothelium, (2) colocalised with caveolin-3, which indicates that FAT/CD36 is in the sarcolemma, or its close vicinity, and (3) abundantly expressed in (or in the close vicinity) of the sarcolemma and intracellular structures of type-1 muscle fibres, and much less abundantly in the sarcolemma of type-2 muscle fibres. One of the antibodies raised against mouse CD36 also detected myosin heavy chain 1, which makes it unsuitable in skeletal muscle research. The fixation (acetone or methanol/acetone) was found to be highly important for the result.