Effect of low-intensity magnetic fields on the development of satellite muscle cells of a newborn rat in the primary culture

The influence of Earth magnetic field shielded down to 0.3 microT and static magnetic field (60-160 microT) on the proliferation and differentiation of satellite muscle cells in the primary culture has been investigated. A stimulatory effect of static magnetic fields on the rate of the formation of massive multinucleated myotubes and an increase in the intracellular calcium concentration ([Ca2+]i) have been detected for magnetic fields of the microtesla range. On the other hand, it was shown that the reduction of earth magnetic fields to 0.3 microT leads to the inhibition of proliferation and differentiation of skeletal muscle cells in the primary culture. Since the formation of contractile myotubes during in vitro experiments is similar to the regeneration of skeletal muscle fibers under muscle damage in vivo, it may be concluded that weak magnetic fields have a strong effect on intracellular processes by influencing all phases of muscle fiber formation. It is necessary to take this fact into consideration when forecasting probable complications of skeletal muscle regeneration during long-term exposure of man to low-intensity magnetic fields and also for the potential use of low static magnetic fields as a tool to recover the affected myogenesis.     

Alternate hydroxylating activities in newborn rat adrenal cells in primary culture

During the course of a study to produce reference compounds, the metabolism of tetrahydrogenated derivatives (ring A reduced) of progesterone, 6 alpha-hydroxyprogesterone, 11-deoxycorticosterone and corticosterone in newborn rat adrenal cells in primary culture was studied. Analysis of the metabolites was made by gas chromatography-mass spectrometry. Most products resulted from the enzymatic reactions of 11 beta-, 18- and 21-hydroxylation, reduction of the 20-oxo group and oxidoreduction of the 3-hydroxyl group. However, unexpected metabolites were produced from the incubation of 3 beta, 5 alpha-tetrahydroprogesterone and 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. They resulted from the 16 alpha-hydroxylation of the precursors and probably from the 15 alpha-, 16 beta- and 17 alpha-hydroxylation of 6 alpha-hydroxy-3 alpha, 5 beta-tetrahydroprogesterone. These hydroxylating activities are weak and were not detected from the endogenous steroidogenesis. They were not detected either from the incubation of exogenous steroids with a 3-oxo-4-ene structure or from steroids with a 21-hydroxyl substituent. They result only from substrates showing diminished or no affinity towards the 11 beta/18- and 21-steroid hydroxylase systems. These unusual hydroxylations could be catalyzed by monooxygenase systems in the endoplasmic reticulum similar to those present in the liver or by the monooxygenase systems specific to steroidogenesis. In particular, the reaction specificity of cytochrome P-450(11) beta could be altered by the presence of a 6 alpha-hydroxyl group in a tetrahydrogenated steroid.     

Proliferation of muscle satellite cells on intact myofibers in culture

Muscle satellite cells are quiescent myogenic stem cells situated between the basal lamina and plasmalemma of mature skeletal muscle fibers. Injury to the fiber triggers the activation and proliferation of satellite cells whose progeny subsequently fuse to form new myotubes during regeneration. In this paper we report the proliferation of satellite cells on single muscle fibers isolated from adult rats and placed in culture. Viable fibers were liberated from muscle with collagenase and purified from non-muscle cells. The fibers were covered with a basal lamina and retained normal morphological characteristics. Each fiber contained two to three satellite cells per 100 myonuclei. Satellite cells showed little proliferative activity in medium with 10% serum but could be induced to enter the cell cycle by chick embryo extract or fibroblast growth factor. Other polypeptide mitogens such as epidermal growth factor, multiplication stimulating activity, and platelet-derived growth factor were ineffective. Mitogen-stimulated satellite cells fused to form new myotubes after 4-5 days in culture. These results imply that satellite cells are under positive growth control since they proliferate in contact with viable mature fibers when stimulated with mitogen. The mature fibers remained viable in culture but did not give rise to mononucleated cells. After several days, however, the fibers began to extend sarcoplasmic sprouts and underwent dedifferentiative changes that led to the formation of multinucleated cells resembling myotubes. These cells reexpressed embryonic isozymes of creatine kinase not made by the mature fibers.     

Uterine smooth muscle cells in primary culture

Summary Smooth muscle cells (SMC) were enzymatically isolated from the myometrium of adult rat and human uteri and grown in primary culture. Cell fine structure and cytoskeletal organization were followed by transmission electron microscopy and cytochemical demonstration of actin filaments, microtubules and intermediate filaments, and initiation of DNA synthesis was investigated by thymidine autoradiography. During the first few days in culture the cells spread out on the substrate and went through a morphological transformation including loss of myofilaments followed by formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Actin filaments aggregated in stress fibers spanning the entire length of the cells and microtubules and intermediate filaments formed a radiating system originating in the juxtanuclear region. In vivo, the SMC contained intermediate filaments reactive for desmin, but as early as the first day of culture expressed vimentin as well. For five days at least, all cells remained positive for both proteins, but the staining for desmin decreased while that for vimentin increased. This structural modification was accompanied by initiation of DNA synthesis, with a peak on day 3 (45–55% labeled nuclei). Subconfluent, growth-arrested primary cultures responded weakly to purified platelet-derived growth factor and serum, and in secondary cultures no response to the mitogenic stimulation was obtained. The observations indicate that uterine SMC cultivated in vitro undergo a transformation from contractile to synthetic phenotype, similar to the transformation described previously for arterial SMC under the same conditions. The proliferative potential of the uterine cells is, however, markedly lower. The findings support the notions that the transition into synthetic phenotype is a necessary but not sufficient requirement for initiation of DNA synthesis in SMC and that visceral and vascular SMC represent separate differentiation pathways.     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of strong static magnetic fields on primary cortical neurons

Intense uniform magnetic fields, such as those used in magnetic resonance imaging (MRI), are thought to exert little influence at the cellular level. Here we report modifications of the signaling cascades in rat cortical neurons cultured for 1 h in magnetic fields of up to 5 Tesla. The activation of c-Jun N-terminal kinase (JNK) increases monotonically with field strength, with a maximal activation of approximately 10% at 5 T, whereas the activation of extra cellular-regulated kinase (ERK) shows a maximum at 0.75 T ( approximately 10%). Since ERK is involved in cellular differentiation, these results indicate a magnetic induction of the signaling events associated with differentiation. However, the cells respond to further increases in field strength by evoking a stress response, since JNK is a stress-activated protein kinase. Three possible mechanisms are discussed and of these, the most plausible is magnetic field induced change in the membrane rest potential, a microscale magnetohydrodynamic effect. This mechanism most likely involves the activation of voltage dependent Ca(2+) channel opening; since intracellular Ca(2+) concentration was also found to be modified by the static magnetic field.     

Cell cycle commitment of rat muscle satellite cells

Satellite cells of adult muscle are quiescent myogenic stem cells that can be induced to enter the cell cycle by an extract of crushed muscle (Bischoff, R. 1986. Dev. Biol. 115:140-147). Here, evidence is presented that the extract acts transiently to commit cells to enter the cell cycle. Satellite cells associated with both live and killed rat myofibers in culture were briefly exposed to muscle extract and the increase in cell number was determined at 48 h in vitro, before the onset of fusion. An 8-12-h exposure to extract with killed, but not live, myofibers was sufficient to produce maximum proliferation of satellite cells. Continuous exposure for over 40 h was needed to sustain proliferation of satellite cells on live myofibers. The role of serum factors was also studied. Neither serum nor muscle extract alone was able to induce proliferation of satellite cells. In the presence of muscle extract, however, satellite cell proliferation was directly proportional to the concentration of serum in the medium. These results suggest that mitogens released from crushed muscle produce long-lasting effects that commit quiescent satellite cells to divide, whereas serum factors are needed to maintain progression through the cell cycle. Contact with a viable myofiber modulates the response of satellite cells to growth factors.     

Bioconversion of 2 alpha-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2 alpha-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11 beta-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6 beta-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2 alpha-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2 beta-epimers of the different metabolites arose principally from the transformation of 2 beta-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11 beta-hydroxylation where the reaction appears stereospecific for the 2 beta-epimer. The 2 alpha-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3 beta-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Nifedipine-activated Ca(2+) permeability in newborn rat cortical collecting duct cells in primary culture

To characterize Ca(2+) transport in newborn rat cortical collecting duct (CCD) cells, we used nifedipine, which in adult rat distal tubules inhibits the intracellular Ca(2+) concentration ([Ca(2+)](i)) increase in response to hormonal activation. We found that the dihydropyridine (DHP) nifedipine (20 microM) produced an increase in [Ca(2+)](i) from 87.6 +/- 3.3 nM to 389.9 +/- 29.0 nM in 65% of the cells. Similar effects of other DHP (BAY K 8644, isradipine) were also observed. Conversely, DHPs did not induce any increase in [Ca(2+)](i) in cells obtained from proximal convoluted tubule. In CCD cells, neither verapamil nor diltiazem induced any rise in [Ca(2+)](i). Experiments in the presence of EGTA showed that external Ca(2+) was required for the nifedipine effect, while lanthanum (20 microM), gadolinium (100 microM), and diltiazem (20 microM) inhibited the effect. Experiments done in the presence of valinomycin resulted in the same nifedipine effect, showing that K(+) channels were not involved in the nifedipine-induced [Ca(2+)](i) rise. H(2)O(2) also triggered [Ca(2+)](i) rise. However, nifedipine-induced [Ca(2+)](i) increase was not affected by protamine. In conclusion, the present results indicate that 1) primary cultures of cells from terminal nephron of newborn rats are a useful tool for investigating Ca(2+) transport mechanisms during growth, and 2) newborn rat CCD cells in primary culture exhibit a new apical nifedipine-activated Ca(2+) channel of capacitive type (either transient receptor potential or leak channel).     

Bioconversion of 16 alpha-hydroxyprogesterone to 16 alpha-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

Using newborn rat adrenal cells in primary culture, 16 alpha-hydroxyprogesterone was bioconverted into numerous 16 alpha-hydroxylated steroids. The method of analysis of these steroids comprised the association of column and thin-layer chromatography to gas chromatography-mass spectrometry in order to obtain the mass spectra of pure compounds. The identified compounds resulted principally from the enzymatic reactions of 21-hydroxylation 11 beta-hydroxylation and reduction of the 20-oxo and 3-oxo-4-ene groups. Minor metabolites resulted from 18-hydroxylation and 6 beta-hydroxylation of the substrate. The metabolism of 16 alpha-hydroxyprogesterone is similar to that of progesterone in the same cell-culture system; however, there are two exceptions. The 21-hydroxylation of 16 alpha-hydroxyprogesterone occurs at a rate similar to that of its 11 beta-hydroxylation, whereas the 21-hydroxylation of progesterone is faster than its 11 beta-hydroxylation. The ratio of 11 beta- to 18-hydroxylation of 16 alpha-hydroxyprogesterone is about 3, whereas the ratio of 11 beta- to 18-hydroxylation of progesterone, 20 alpha-dihydroxyprogesterone and DOC is between 1./ and 2. It is most likely the rate of 18-hydroxylation which is decreased by the hydroxyl group at C-16. The use of adrenal cell cultures is a practical, simple method for the preparation of a variety of 16 alpha-hydroxylated steroids from a single substrate. Its adaptation to the production of important amounts of 16 alpha-hydroxylated corticosteroids will permit the study of their biological activity.     

The role of ACTH in determining the metabolic pathways of deoxycorticosterone by newborn rat adrenal cells in primary culture

The metabolism of deoxycorticosterone (DOC) by newborn rat adrenal cells in primary culture at various times after culture, with and without ACTH, was studied. After 5 days in culture before addition of ACTH, the main products of the metabolism of DOC were corticosterone and 18-hydroxy-11-deoxycorticosterone in a 2:1 ratio. Smaller amounts of 20 alpha-dihydrocorticosterone and 18-hydroxycorticosterone were also found. No reduced metabolites of DOC were detected. Without ACTH the conversion of DOC to corticosterone and 18-hydroxyDOC declined rapidly. After 13 days in culture, this conversion accounted for only half the metabolites. The reductive metabolism of DOC which yields products reduced at 20 alpha and/or 3 alpha/beta and 5 alpha accounted for the other half. When ACTH (22 mU/ml) was added to the culture daily for several weeks, the primary metabolism of DOC remained that of 11 beta- and 18-hydroxylation yielding corticosterone and 18-hydroxyDOC. A minor reductive metabolism was found. Both cultures produced 6 beta-hydroxyDOC. These results demonstrate that ACTH is needed to maintain the efficiency of the 11 beta/18-hydroxylating system. They also show that ACTH controls the type of metabolism predominant in the rat adrenal cell and may be responsible for the balance between the biosynthesis of glucocorticoids and their reductive catabolism in the fasciculata zone of the adrenal gland.     

Observations of satellite cells in rat skeletal muscle incubated in vitro

Summary Satellite cells were studied in the peripheral fibres from isolated rat muscles, which had been incubated for various periods of time. The cells were in an activated state with prominent organelles and increased cytoplasm visible. Mitosis of some satellite cells was occasionally observed. It is suggested that when incubated muscle preparations are used as models for physiological systems in vivo, the state of the satellite cell population should be taken into consideration.     

Bioconversion of 2α-hydroxyprogesterone to 2-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture

The bioconversion of 2α-hydroxyprogesterone into 2-hydroxylated steroids was accomplished using newborn rat adrenal cells in primary culture. The products were purified using column and thin-layer chromatography, and identified by GC-MS. They resulted principally from the enzymatic reactions of 21-hydroxylation, 11β-hydroxylation, reduction of 20-oxo and 3-oxo groups, and epimerization of the substrate. In addition, minor metabolites resulted from 18-hydroxylation, 6β-hydroxylation and reduction of the 3-oxo-4-ene group. The identification of these compounds allowed us to conclude that the metabolism of 2α-hydroxyprogesterone is similar to that of progesterone in this cellular system. Assuming that the 2β-epimers of the different metabolites arose principally from the transformation of 2β-hydroxyprogesterone, the specificity of the various enzyme systems seems to be similar for both epimers except in the case of the 11β-hydroxylation where the reaction appears stereospecific for the 2β-epimer. The 2α-hydroxyl group on ring A seems to favor the reduction of the 3-oxo group and it does this stereospecifically to the 3β-structure. The epimerization of the substrate, which is most likely enzymatically induced, is the first example of steroid epimerization reported in the adrenal. This is a practical preparative method for synthesizing a variety of steroids hydroxylated at C-2 from a single substrate and could be adjusted to the production of important quantities of 2-hydroxylated metabolites of corticosteroids.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

Precocious in vitro development of satellite cells from dystrophic chicken muscle

Satellite cells, liberated from pectoral muscle of juvenile dystrophic chickens by sequential treatment with collagenase, hyaluronidase, and trypsin and preplated to remove fibroblasts and cultured on gelatin proliferated rapidly, fused and formed confluent muscle cultures within 6 d in vitro with minimal contamination by fibroblasts. When identical isolation and culturing techniques were applied to muscle from age-matched normal chickens proliferation and differentiation were slower, contamination with fibroblasts was much greater, and only a small number of myotubes were formed. After injection of the myotoxic anesthetic marcaine into normal pectoral muscle for 5 consecutive days, myotube formation was accelerated in satellite cell cultures, but the rate of differentiation was not as rapid as that occurring in cells from dystrophic muscle.