Characterization of satellite cells derived from chickens with the low score normal (LSN) muscle weakness

Myogenic satellite cell clones were established from the pectoralis major muscle of chickens with the low score normal (LSN) muscle weakness and controls. The percentage of cells which attached to substrata and began to proliferate was higher for the control line than the LSN line (55% vs 30%). Furthermore, of those clones which initiated growth, 63% of the control cells and only 32% of the LSN cells proliferated to confluence in 25 cm2 tissue culture flasks. Proliferation rates were significantly lower with LSN satellite cells than with controls (p < 0.05). LSN satellite cells were less responsive to the mitogenic effects of chicken serum (p < 0.05) and differentiation rates were lower compared with controls (p < 0.05). There was a greater (p < 0.05) number of insulin-like growth factor receptors on LSN satellite cells compared with controls. The IGF receptor binding affinities (Kds) between the two cell lines were similar (p > 0.05). The results suggest that a defect in satellite cell physiology may contribute to the skeletal muscle weakness seen in the LSN line.     

Expression of a voltage-dependent potassium current precedes fusion of human muscle satellite cells (myoblasts)

Using the whole-cell recording patch clamp technique in clonal cultures of human muscle satellite cells (SC), we studied a voltage-gated potassium current analogous to the delayed rectifier current (IKdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed IKdr. In contrast, IKdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum-free medium (SFM) and within 24 h in differentiating medium. We believe that IKdr may be a characteristic feature of fusion-component SC and that it may be involved in the fusion process for the following reasons: 1) after the transfer in differentiating medium, cultures of SC in which the expression of IKdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; 2) in the latter "naive" SC, IKdr was expressed during the first day in differentiating medium, before SC began to fuse; 3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express IKdr; 4) TEA, at a concentration of 3 mM, reduces the amplitude of IKdr by 55% and the fusion index by 55-67%.     

Quantitative histochemical evaluation of normal human skeletal muscle

Skeletal muscle tissue was obtained by open biopsy from the vastus lateralis and peroneus brevis muscles from 12 and 16 healthy paid volunteers, respectively. Frozen sections were examined with standard histochemical methods. Central nuclei, small and large angular fibers, and small round fibers were the most common "abnormalities" present. The number of fibers of these types were quantified, along with other more rare deviations from normal morphology. Several of the abnormalities were more common in the peroneus brevis than in the vastus lateralis.     

Respiratory-related pharyngeal constrictor muscle activity in normal human adults

Electromyographic activity of the superior, middle, and inferior pharyngeal constrictor (PC) muscles was examined in 10 normal adult humans during wakefulness and sleep. Wire electrodes were inserted close to the midline of the posterior pharyngeal wall at the level of the soft palate (superior PC), tip of the epiglottis (middle PC), and corniculate tubercle (inferior PC). In general, the three PC muscles exhibited similar patterns of activation. The PCs were activated during swallows, repetitive "pa" sounds, changes in head position, and the last portions of slow inspiratory and expiratory vital capacity maneuvers. Respiratory-related PC activity was infrequent during quiet breathing during wakefulness; variable and inconsistent phasic activation in expiration in one or more of the PCs was present in seven of the 10 subjects, particularly after a swallow. Progressive hyperoxic hypercapnia and progressive isocapnic hypoxia were associated with recruitment of phasic PC activity, which was predominantly expiratory; however, variable discharge patterns were observed within a given muscle and a given subject. When phasic PC activity was present, preactivation during late inspiration was frequently observed. PC activity was absent in NREM sleep and exhibited sporadic, nonrespiratory-related bursts of activity during REM sleep. Passively induced hypocapnic hyperventilation in NREM sleep was not associated with PC activation. The results indicate that the PCs have very similar patterns of activation and exhibit phasic expiratory activity during relatively high ventilatory output states in wakefulness.     

Cultures of normal human mammary cells

This paper describes a method of obtaining epithelial cells from large quantities of normal human breast tissue and the response of these cells in culture to lactogenic hormones. Suspensions of single cells and clusters of cells resembling normal ductal and alveolar structures were obtained by mechanical disaggregation and subsequent (3h) incubation of tissue fragments in 0.5 mg/ml collagenase. Cells rapidly attached to glass or plastic surfaces within 48 h and grew to form large colonies which maintained their epithelial appearance throughout 2 months of observation. Cell cycling as monitored by DNA synthesis was enhanced by insulin, hydrocortisone, or ovine prolactin (in concentrations of 5.0mug/ml each) at respectively 2,3 and 5 days of incubation. These results were observed in cultures derived from 3 premenopause samples of mammary tissue maintained in medium with 1% fetal calf serum. Prolactin at a concentration of 5 mug/ml induced phosphoprotein synthesis 8-fold over control values. In addition, prolactin induced morphological changes in cells including the development of distended endoplasmic reticulum, large microvilli, and the deposition of glycogen granules. These initial results led to the tentative conclusion that prolactin was sufficient to initiate some of the characteristics in cultured cells normally associated with lactating tissues.     

Immunolocalization of leukemia inhibitory factor in normal and denervated human muscle

The cytokine leukemia inhibitory factor (LIF) stimulates myoblast proliferation in vitro and vivo and is neurotrophic for motor neurons. In experimentally reinnervated muscle, exogenous LIF application increases muscle mass through myofiber hypertrophy. The goal of this study was to evaluate possible sources of endogenous LIF in human muscle, and whether LIF immunoreactivity (-IR) was detectable in specific myofiber types and/or re-expressed in human denervated muscle. Our study shows that LIF-IR is constitutively detectable in type I myofibers of normal human muscle. In acute and chronically denervated and reinnervated human muscle, LIF-IR is found in all type I myofibers and in addition in some atrophic and almost all angulated atrophic type II myofibers.     

Detection of normal and chimeric nucleophosmin in human cells

In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RARalpha and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.     

Control of glycogen synthesis in cultured human muscle cells

The regulation of glycogen synthesis and associated enzymes was studied in human myoblasts and myotubes maintained in culture. Both epidermal growth factor (EGF) and insulin stimulated glycogen synthesis approximately 2-fold, this stimulation being accompanied by a rapid and stable activation of the controlling enzyme glycogen synthase (GS). EGF also caused inhibition of glycogen synthase kinase 3 (GSK-3) and activation of the alpha isoform of protein kinase B (PKB) with the time-course and magnitude of its effects being similar to those induced by insulin. An inhibitor of the mitogen-activated protein (MAP) kinase pathway did not prevent stimulation of GS by EGF, suggesting that this pathway is not essential for the effect. A partial decrease in the fold activation of GS was, however, observed when p70(S6k) activation was blocked with rapamycin, suggesting a contribution of this pathway to the control of GS by either hormone. Wortmannin, a selective inhibitor of phosphatidylinositol 3'-kinase (PI-3 kinase) completely blocked the effects of both EGF and insulin in these cells. These results demonstrate that EGF, like insulin, activates glycogen synthesis in muscle, acting principally via the PKB/GSK-3 pathway but with a contribution from a rapamycin-sensitive component that lies downstream of PI-3 kinase.     

Fibroblast growth factor promotes recruitment of skeletal muscle satellite cells in young and old rats

Although the role of satellite cells in muscle growth and repair is well recognized, understanding of the molecular events that accompany their activation and proliferation is limited. In this study, we used the single myofiber culture model for comparing the proliferative dynamics of satellite cells from growing (3-week-old), young adult (8- to 10-week-old), and old (9- to 11-month-old) rats. In these fiber cultures, the satellite cells are maintained in their in situ position underneath the fiber basement membrane. We first demonstrate that the cytoplasm of fiber-associated satellite cells can be monitored with an antibody against the extracellular signal regulated kinases 1 and 2 (ERK1 and ERK2), which belong to the mitogen-activated protein kinase (MAPK) superfamily. With this immunocytological marker, we show that the satellite cells from all three age groups first proliferate and express PCNA and MyoD, and subsequently, about 24 hr later, exit the PCNA+/MyoD+ state and become positive for myogenin. For all three age groups, fibroblast growth factor 2 (FGF2) enhances by about twofold the number of satellite cells that are capable of proliferation, as determined by monitoring the number of cells that transit from the MAPK+ phenotype to the PCNA+/MAPK+ or MyoD+/MAPK+ phenotype. Furthermore, contrary to the commonly accepted convention, we show that in the fiber cultures FGF2 does not suppress the subsequent transition of the proliferating cells into the myogenin+ compartment. Although myogenesis of satellite cells from growing, young adult, and old rats follows a similar program, two distinctive features were identified for satellite cells in fiber cultures from the old rats. First, a large number of MAPK+ cells do not appear to enter the MyoD-myogenin expression program. Second, the maximal number of proliferating satellite cells is attained a day later than in cultures from the young adults. This apparent "lag" in proliferation was not affected by hepatocyte growth factor (HGF), which has been implicated in accelerating the first round of satellite cell proliferation. HGF and FGF2 were equally efficient in promoting proliferation of satellite cells in fibers from old rats. Collectively, the investigation suggests that FGF plays a critical role in the recruitment of satellite cells into proliferation.     

The effect of radiographic contrast media on human vascular smooth muscle cells

The relation between intravascular radiographic contrast media (RCM) and myointimal hyperplasia after percutaneous transluminal angioplasty is not known. We have investigated the cytotoxic effects of RCM on human vascular smooth muscle cells (VSMCs) and their effect on the growth of these cells. The cytotoxic effects of RCM were studied using human VSMCs. The cells after being grown to confluency were exposed for 60 min to 250 mgI ml-1 of diatrizoate, ioxaglate, iopromide, iotrolan and saturated mannitol solutions. The control group was treated with only 15% fetal calf serum (FCS) containing medium. The viability of the cells was examined using the trypan blue exclusion test. The effect of RCM on growth was assessed by exposing the VSMCs after growth arrest, for either 15 or 60 min to 250 mgI ml-1 of diatrozoate, ioxaglate, iopromide, iotrolan and saturated mannitol solution. There was no significant change in the viability of the VSMCs after 60 min exposure to iopromide, iotrolan, saturated mannitol solution, and after 15 min exposure to diatrizoate or ioxaglate. After exposure to diatrizoate or ioxaglate for 60 min, 16.5 +/- 2.2% or 9.2 +/- 2.6% dead cells were found, respectively (p < 0.05 versus control). In the growth assay of VSMCs, diatrizoate, ioxaglate and saturated mannitol solutions reduced the growth rate (p < 0.05 versus control). No significant change was observed with iopromide and iotrolan. In conclusion, ionic RCM have cytotoxic and cytostatic effects on VSMCs while non-ionic media have no effects. There is no direct stimulatory effect of contrast media on the growth of VSMCs. The cytotoxic and cytostatic effects of contrast media seems to be both osmolality and chemotoxicity dependent. Low osmolar non-ionic RCM are not likely to contribute to the mechanisms responsible for myointimal hyperplasia after angioplasty.     

Histamine induced contraction of human ciliary muscle cells

In cultured human ciliary muscle cells we previously showed that histamine, via an H1 receptor, stimulates the production of inositol phosphates and mobilization of intracellular calcium. We further investigated in this study whether histamine would cause contraction of human ciliary muscle cells. Photomicrographs were taken of the ciliary muscle cells before and after exposure to histamine. Cross sectional surface area of the cells was quantified using image analysis software. A decrease in cross sectional surface area was interpreted as an indication of cell contraction. The results of this study indicated that histamine (10(-6) M-10(-4) M) caused contraction of human ciliary muscle cells in a concentration-dependent fashion. The effect of histamine was mediated by the H1 receptor subtype since the histamine effect was antagonized by 10(-6) M chlorphentramine (an H1 receptor subtype selective antagonist) but not by 10(-6) M cimetidine (H2 antagonist) or thioperamide (H3 antagonist). The phospholipase C (PLC) inhibitor, U73122 (10(-6) M) and the intracellular calcium store depleting agent thapsigargin (10(-6) M) both prevented the histamine induced contraction, demonstrating that the activation of PLC and the intracellular calcium release were the key steps necessary for contraction. Our data indicate that in ciliary muscle cells, histamine, via an H1 receptor, activates PLC and increases intracellular calcium, which subsequently causes contraction of the cells.     

Uptake of triiodothyronine into cultured human muscle cells

The uptake of T3 was measured in cultured human muscle cells at 37 degrees C and pH 7.4 in a medium containing albumin and glucose. The initial up]take increased linearly when the total T3 concentration was varied from 10(-9) to 10(-4) M. At prolonged incubation time the uptake decreased to virtually zero in about 30 min. These data indicate a rapid passive transport mechanism of T3 and a fast equilibration of the cellular T3 with the surrounding medium. In agreement with these conclusions the efflux of T3 was rapid and the initial uptake was not altered by pre-incubation in a T3-containing medium.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca2+ ([Ca2+]i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca2+, mobilised Ca2+ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca2+ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow "pacemaker" and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca2+]i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca(2+)-free solution, but could be returned to a higher level if the cell was exposed to external Ca2+. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca(2+)-containing medium reactivated responses. In the absence of external Ca2+, continuous application of histamine activated a series of transient increases in intracellular Ca2+, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca2+ stores and their sensitivity to cytoplasmic Ca2+ and intrasarcoplasmic reticulum Ca2+.     

Arachidonic acid-induced calcium signalling in human airway smooth muscle cells

Until now computer-assisted parasite identification was based on database applications requiring data specification on an individual basis, thus limiting the ability of the system to handle rule-based knowledge as humans are used to do. A new Expert PArasite IdentificatiON (EPAION: Greek term for expert) system was developed to serve as an interface between the database and the user, where the database is a repository for bionomic and morphological facts about the parasites for the expert system. The system was developed by using a logic-based computer language which allows the definition of rules and facts to assist the creation of queries to the database. The components of the system are the knowledge base, the multimedia data base, the inference mechanism, and the graphical user interface. The operational modules of the system are the Parasite Identifier and the system Utilities. This expert system facilitates knowledge incorporation in a manner simulating the natural mental process, thus allowing the checking of the accuracy of the information that the user feeds to the computer and the creation of intelligent queries to the database. These characteristics accelerate focusing and optimize the parasite identification scheme regardless of the user's profile of competency.     

Na-K-Cl cotransport in normal and glaucomatous human trabecular meshwork cells

PURPOSE: Previous results from this laboratory showed that intracellular volume of trabecular meshwork (TM) cells is regulated by the Na-K-Cl cotransport system. Other studies suggest that TM cell volume, in turn, is a determinant of permeability across the TM. Given that a decrease in outflow facility across the TM is thought to be the primary cause of elevated intraocular pressure in primary open-angle glaucoma, the present study was conducted to investigate the possibility that Na-K-Cl cotransport function may be altered in glaucomatous TM cells compared with normal TM cells. METHODS: Normal and glaucomatous human TM cells were cultured from donor eyes and trabeculectomy specimens, respectively. Trabecular meshwork cell monolayers were evaluated for Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx using 86Rb as a tracer for K. Cotransporter protein expression was determined by western blot analysis, and intracellular volume was determined radioisotopically using [14C]urea and [14C]sucrose as markers of total and extracellular water space, respectively. RESULTS: Na-K-Cl cotransport activity of glaucomatous TM cells was found to be reduced by 32% +/- 2% compared with that of normal TM cells, whereas western blot analyses showed that cotransporter protein expression in glaucomatous TM cells was reduced by 64% +/- 14% compared with expression in normal TM cells. Also, exposure of normal TM cells to 10 microM norepinephrine or 50 microM 8-bromo-3',5'-cyclic adenosine monophosphate was found to diminish Na-K-Cl cotransport activity, whereas these agents were without effect on glaucomatous TM cell cotransport. Finally, resting cell volume of glaucomatous TM cells was found to be increased compared with that of normal TM cells, whereas intracellular volume of both cell types was reduced after exposure to 10 microM benzmetanide or 10 microM bumetanide. CONCLUSIONS: These findings indicate that Na-K-Cl cotransport function and regulation are altered in glaucomatous TM cells compared with that of normal TM cells. However, the observation that cell volume of glaucomatous TM cells is greater than that of normal TM cells, despite reduced Na-K-Cl cotransport activity, suggests that other volume-regulatory ion flux pathways may be involved in the reduced outflow of glaucoma.     

Satellite cells and muscle regeneration in diseased human skeletal muscles

By virtue of the lanthanum nitrate staining technique applied to biopsied muscle we are able to demonstrate interaction between satellite cells and parent myofibers, as well as development of premyocytes from activated satellite cells. The process of regeneration in diseased muscle appears to differ from that described in experimental myogenesis. Transformation of activated satellite cells to two types of premyocytes in the process of muscle regeneration seems to rely primarily on the state of innervation and recovery rate of the parent cell after injury. Activated satellite cells are characterized morphologically by proliferation of caveolae, first on the parent fiber side, and early T-tubule and myofilament formation and central displacement. In diseased human muscle the satellite cells appear to play significant roles in muscle regeneration both as a source of reinforcement for failing metabolism in the parent cell and as potential replacements for the necrotic segment of the parent cell. This study also demonstrates that the satellite cells are capable of developing into independent myocytes which may fuse with or replace the parent cell, dependent upon the type and extent of the injury sustained. Abnormal fusion among premyocytes or with their parent fiber, resulting in formation of split-or ring-fibers, becomes conceivable when both innervation and recovery from the injury of the parent cell are delayed. Thus, myotube formation, characteristic of usual myogenesis, seldom takes place in the regenerative process instituted by satellite cells in diseased human skeletal muscles.