Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.     

Tropomyosin heterogeneity in human cells

Tropomyosin preparations from human platelets, human peripheral blood leukocytes from normal individuals and from a patient with chronic lymphocytic leukemia, human lymphoblastoid cells (GM607), human epithelial cells, and human skin fibroblasts have all been found to contain more than one protein when analyzed by two-dimensional gel electrophoresis. Although the lymphoid cell preparations consistently contain two proteins of almost identical molecular weight (Mr = 30,000), the platelet, epithelial cell, and fibroblast preparations contain two or more major proteins with molecular weights between 31,000 and 36,000, in addition to a major protein at 30,000. All of these proteins have characteristics in common with tropomyosin including slightly acidic isoelectric point (approximately pH 4), stability to heat and organic solvents, association with the cytoskeleton, and reactivity with antibody against skeletal muscle tropomyosin. The nonmuscle tropomyosin-like proteins were compared with tropomysins from human skeletal, cardiac, and smooth muscle by peptide mapping after partial proteolysis. The results showed one of the non-muscle proteins to be identical to the major smooth muscle tropomyosin in human uterus (myometrium) and another to be similar but not identical to skeletal muscle alpha-tropomyosin. The remainder of the proteins with tropomyosin characteristics was unique to non-muscle cells. In all, nine distinct human proteins with characteristics of tropomyosin are described. Charge variants of two of these proteins have been described previously.     

Paradoxical effects of glucocorticoids on regulation of plasminogen activator activity: Mediation by glucocorticoid receptors

Dexamethasone, a synthetic glucocorticoid, decreases the plasminogen activator (PA) activity of HTC rat hepatoma cells in tissue culture. Paradoxically, dexamethasone enhances the cyclic nucleotide stimulation of PA activity in these cells 2–4-fold. In this report, we investigated whether this paradoxical glucocorticoid effect is mediated by the same proximal events which mediate such direct regulatory actions of glucocorticoids as the induction of tyrosine aminotransferase activity. We compared the concentration-dependences for several classes of steroids, previously classified as full agonists, partial agonists, antagonists or inactive steroids with respect to induction of the transaminase, for both enhancement of cyclic nucleotide stimulation of PA activity and induction of tyrosine aminotransferase activity in parallel cultures. The full agonists dexamethasone and cortisol, the partial agonists deoxycorticosterone and 11β-hydroxyprogesterone, the inactive steroid tetrahydrocortisol, and the antagonist 17α-methyltestosterone exhibited similar potencies with respect to both phenomena. Furthermore, when cells were incubated with both dexamethasone and 17α-methyltestosterone, the latter blocked enhancement by dexamethasone in a concentration-dependent fashion. We conclude that glucocorticoid enhancement of cyclic nucleotide stimulation of PA activity is mediated by the same glucocorticoid receptors which mediate direct regulatory effects.     

Effects of 5-bromo-2''-deoxyuridine on beating heart cell cultures from neonatal hamsters.

1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.     

Heart ventricular cells of neonatal rats in a culture: DNA synthesis, ultrastructure and localization of atrial natriuretic peptide

We determined the optimal conditions suitable for expanding cardiac cells in vitro for their future use in experimental transplantation into injured myocardium of adult animals. Ventricular cardiac cells were isolated enzymatically from 2-3 day-old rats and cultured at different cell densities within 5-7 days to 4 weeks. Mixed cultures of muscle and non-muscle cells were examined by light autoradiography, electron microscopy, and immunogold method. The best results were obtained at a density of 3 x 10(5) cells/ml in the medium, consisting of 90% DMEM and 10% fetal calf serum, during 5-7 days of cultivation. In such cultures myocytes made 62.5 +/- 7.9%. After a 24 h incubation with 3H-thymidine, 22.0 +/- 2.2% of myocytes were labeled. Muscle cells contact with each other and with non-muscle cells, contain myofibrils, contract and display atrial natriuretic peptide (ANP)-like immunoreactivity.     

Studies of adult amphibian heart cells in Vitro: DNA synthesis and mitosis

The ventricle of the adult newt heart was excised and cut into several pieces of approximately 0.5 – 1.0 mm. These heart pieces were then cultured for 60 days at 25 °C in a modified Leibovitz medium (L-15). Approximately 37% of the explants were attached to the substrate and more than 33% of the attached explants and approximately 15% of the unattached explants established pulsation rates which ranged 3–67 beats/min. The explants were labeled with 1 μCi/ml of ³H-thymidine for 24 hr at 7, 15, 21, 30, 45 and 60 days of culture initiation, and processed for electron microscopic autoradiography. The examination of the autoradiograms revealed that as the culture continued, the cardiac muscle cells altered their morphology, resembling embryonic cardiac muscle cells. These altered muscle cells were termed dedifferentiated cardiac muscle cells. The number of these dedifferentiated cells increased over the period of culture, showing 10.3–94% dedifferentiated cells after 7–60 days of culture respectively. DNA synthesis and mitosis were observed in the dedifferentiated cardiac muscle cells, apart from the non-muscle cells. The quantitation of the autoradiograms revealed that the number of labeled nuclei in the cardiac muscle cells gradually increased over the period of culture, and a maximum number of labeled cardiac muscle cells (30%) was observed in the third week. The peak was followed by a decline in the eighth week which exhibited 1.5 % labeled cardiac muscle cells. The trend of mitosis was similar to that of DNA synthesis. The maximum number of mitotic figures (9%) was observed in the third week of culture, which was followed by a decline and finally absent in the eighth week. The cardiac non-muscle cells, mostly fibroblasts and endothelial cells, also showed incorporation of ³H-thymidine in their nuclei. The number of labeled non-muscle cells nuclei and the mitotic index were highest (61 and 15% respectively) in the first week of culture, but then they decreased gradually over the eight-week period in culture. This study provides evidence for the first time that the adult amphibian cardiac myocytes can undergo DNA synthesis and mitosis when explanted and cultured. The significance of this cell replication is discussed.     

Intracellular proteolysis in rat cardiac and skeletal muscle cells in culture.

Treatment of adult rats with dexamethasone resulted in an increase in cardiac muscle weight but a decrease in skeletal muscle weight. The different response of skeletal and cardiac muscles to the glucocorticoid was also reflected by a dexamethasone-induced enhancement of myofibrillar protease activity in the gastrocnemius muscle and an inhibition of a similar proteolytic activity in the heart. Newborn rats also exhibit the same, tissue-specific response to the glucocorticoid hormone. Consequently, the difference between cardiac and skeletal muscle responsiveness to conditions of wasting was investigated in culture. Average rates of degradation of intracellular proteins were determined in cultured cells derived from rat skeletal and cardiac muscle by following the release of radioactivity from cells prelabelled with ¹⁴C-phenylalanine. The release of label into the TCA soluble medium as measured during 12 hours of incubation, conformed to a first-order reaction and both cell types were found to degrade intracellular proteins at a similar rate. After 12 hours of incubation in a complete Ham F-10 medium supplemented with serum approximately 18% of total cellular protein was degraded. Incubation in a minimal medium or serum-deprivation enhanced the average rate of proteolysis to a value of 29% degradation at 12 hours indicating that intracellular proteolysis in these cells is responding to nutritional deprivation by increased activity. However, addition of glucose (22.2 nM) or dexamethasone (10^?6M) to the incubation medium failed to affect the rate of net protein degradation. Under no experimental condition could a difference be found between the proteolytic response of skeletal muscle cells to that of cardiac muscle cells and both cell types displayed similar changes in rates of protein degradation under various nutritional and hormonal conditions in culture. Thus, protein sparing in the heart of intact animals under catabolic conditions which enhance protein loss in skeletal muscle can probably not be ascribed to intrinsic differences in the direct response of cellular proteases to the tested hormones and nutrients. Rather, an extracellular factor(s) is apparently required for induction of the differential response of these tissues in the intact animal to protein wasting conditions. Alternatively, cells in culture might have lost the property of differential degradative response which operates in vivo.     

Macrolactonolides: A novel class of anti-inflammatory compounds

A new concept in design of safe glucocorticoid therapy was introduced by conjugating potent glucocorticoid steroids with macrolides (macrolactonolides). These compounds were synthesized from various steroid 17β-carboxylic acids and 9a-N-(3-aminoalkyl) derivatives of 9-deokso-9a-aza-9a-homoeritromicin A and 3-descladinosyl-9-deokso-9a-aza-9a-homoeritromicin A using stable alkyl chain. Combining property of macrolides to preferentially accumulate in immune cells, especially in phagocyte cells, with anti-inflammatory activity of classic steroids, we designed molecules which showed good anti-inflammatory activity in ovalbumin (OVA) induced asthma in rats. The synthesis, in vitro and in vivo anti-inflammatory activity of this novel class of compounds are described.     

The Influence of Immobilization Stress on Cardiac Protein Synthesis. A Possible Regulatory Role for Glucocorticolds

Abstract

High serum corticosterone levels and transient depletion of cytosolic glucocorticoid receptor binding capacity were observed in rat cardiac muscle following immobilization stress. To evaluate the effect of this treatment on the protein synthetic capacity, biologically active polyribosomes were used to direct the in vitro synthesis of polypeptides in the rabbit reticulocyte lysate. The results of these experiments indicate that the template activity of several messenger RNAs coding for major myofibrillar proteins was increased. This change in protein synthetic activity was not observed in adrenalectomized animals. Collectively, these results suggest that glucocorticoids are involved in the regulation of cardiac protein biosynthesis.     

Glucocorticoids Modify Differentially Dopamine- and Prostaglandin E1-Mediated Cyclic AMP Formation by the Cultured Human Astrocytoma Clone D384

The effects of steroid hormones on the cyclic AMP responses to stimulation of human astrocytoma cells (D384) by dopamine, prostaglandin E1 (PGE1), and isoprenaline were investigated. Incubation of D384 cells with dexamethasone resulted in a potentiation of the PGE1 and isoprenaline responses and a marked attenuation of the dopamine response. The time courses of the effects of dexamethasone on dopamine and PGE1 responses were similar, requiring long-term (at least 18 h) incubation of cells with the steroid. Concentration-response curves of dexamethasone effects on dopamine and PGE1 responses yielded similar Ka apparent values, suggesting a common mechanism. Cycloheximide, a protein synthesis inhibitor, prevented the effects of dexamethasone. Only steroids with glucocorticoid activity reproduced the dexamethasone effects. Direct stimulation of Gs with 5-guanylylimidodiphosphate and adenylate cyclase with forskolin revealed no significant differences in their activities in dexamethasone-treated and untreated cells. Furthermore, a comparison of the dopamine and PGE1 concentration-response curves obtained from dexamethasone-treated and untreated cells suggested that the affinity of the receptors for their agonists remained unchanged. These results suggest that glucocorticoids may alter protein synthesis and thereby the number of receptors expressed by D384 cells.     

Synthesis of heat-shock proteins by cells undergoing myogenesis

Subjecting 24-h-old cultures of quail myoblasts to incubation at an elevated temperature causes the pattern of protein synthesis to shift from the production of a broad spectrum of different proteins to the enhanced synthesis of a small number of heat-shock proteins. The synthesis of four major heat-induced polypeptides with Mrs of 88,000, 82,000, 64,000 and 25,000 achieve levels comparable to that of the major structural protein, actin. Two-dimensional electrophoretic separation and fluorographic analysis of these polypeptides establish that those with Mrs of 94,000, 88,000, 82,000, and 64,000 and pIs of 5.1, 5.2, 5.2, and 5.4, respectively, are synthesized by heat-shocked as well as by control (albeit not as intense) cultures. However, the synthesis of polypeptides with Mrs of 94,000, 64,000, and 25,000 and pI's of 5.2, 5.8, and 5.4, respectively, is detectable only in myoblasts shifted to a higher temperature. Recovery of heat-shocked myoblasts, to a normal preinduction pattern of polypeptide synthesis, takes approximately 8 h. Similar studies, completed in older, more differentiated myogenic cells, demonstrated that as cells progress through myogenesis their ability to respond to a similar temperature shift is diminished. The synthesis of some myoblastlike heat-shock proteins by fusing of cells or by myotubes requires that they be maintained at an elevated temperature at least twice as long as myoblasts. This observation and the demonstration that heat-shocked myotubes do not synthesize detectable levels of the 25,000-dalton polypeptide found in heat-shocked myoblasts, suggest that the synthetic response of myogenic cells to heat shock is dependent on the differentiative state of these cells.     

Steroid binding activity is retained in a 16-kDa fragment of the steroid binding domain of rat glucocorticoid receptors

The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites.     

Induction of cytochrome P-450 by glucocorticoids in rat liver. II. Evidence that glucocorticoids regulate induction of cytochrome P-450 by a nonclassical receptor mechanism

In the companion report we used primary cultures of adult rat hepatocytes to demonstrate that glucocorticoids comprise a "class" of compounds that stimulate de novo synthesis of a form of cytochrome P-450 (P450PCN) indistinguishable from that induced by the nonhormonal steroid pregnenolone 16 alpha-carbonitrile (PCN). Because induction of P450PCN is stereospecific for glucocorticoids and is dependent on the concentration of and the length of exposure to steroids it seemed possible that P450PCN represented another of the many genes whose expression is coordinately regulated by glucocorticoids bound to their specific cytoplasmic receptor and translocated into the nucleus. However, in cultured hepatocytes treated with glucocorticoids, synthesis of P450PCN failed to parallel synthesis of a typical glucocorticoid-responsive liver function, tyrosine aminotransferase, in the time course of induction, in the concentrations of glucocorticoids required for half-maximal induction, and in the order of effective steroids ranked by potency. Indeed, two moderately potent inducers of P450PCN either failed to induce tyrosine aminotransferase (spironolactone) or actually antagonized induction of tyrosine aminotransferase synthesis by glucocorticoids (PCN). Moreover, in the same cultures in which glucocorticoid induction of tyrosine aminotransferase was blocked by the presence of PCN or other previously identified antiglucocorticoids, synthesis of P450PCN was actually enhanced. We conclude that synthesis of P450PCN is a specific glucocorticoid-responsive liver function evoked by a novel mechanism readily distinguishable from the classic glucocorticoid receptor pathway.     

Autoradiographic study of protein synthesis in the muscle and interstitial elements of the myocardium during development of compensatory heart hyperfunction

In the emergency stage of heart compensatory hypertrophy induced by constriction of the abdominal aorta, quantitative autoradiographic localization of newly synthesized proteins and RNA was studied in the left ventricular myocardium of adult rats 1 and 3 hours after the injection of 3H-leucine and 3H-uridine, respectively. The animals were sacrificed 1, 5 and 10 days after the operation. The amount of the autoradiographic grains was measured separately for muscle and interstitial components of the myocardium. A substantial increase in protein synthesis as regards muscle and non-muscle components was recorded only on day 10 of the experiment. At the same time incorporation of amino acids into protein of muscle and interstitial components was found to be higher by 42 and 60%, respectively. The labeling of RNA in muscle cells was similar to that of protein. In interstitial cells, the content of labeled RNA consistently rose throughout the whole experiment.     

Turnover of myosin heavy and light chains in cultured embryonic chick cardiac and skeletal muscle

The relative rates of synthesis and breakdown of myosin heavy and light chains were studied in primary cell cultures of embryonic chick cardiac and skeletal muscle. Measurements were made after 4 days in culture, at which time both skeletal and cardiac cultures were differentiated and contracted spontaneously. Following a 4-hr pulse of radioactive leucine, myosin and its heavy and light chains were extracted to 90% or greater purity and the specific activities of the proteins were determined. In cardiac muscle, myosin heavy chains were synthesized approximately 1.6 times the rate of myosin light chains, and in skeletal muscle, heavy chains were synthesized at approximately 1.4 times the rate of light chains. Relative rates of degradation of muscle proteins were determined using a dual-isotope technique. In general, the soluble and myofibrillar proteins of both types of muscle had decay rates proportional to their molecular weights (larger proteins generally had higher decay rates) based on analyses utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A notable exception to this general rule was myosin heavy chains, which had decay rates only slightly higher than the myosin light chains. Direct measurements on purified proteins indicated that the heavy chains of myosin were turning over at a slightly greater rate (approximately 20%) than the myosin light chains in both cardiac and skeletal muscle. The reasons for the apparent discrepancy between these measurements of myosin heavy and light chain synthesis and degradation are discussed.     

Glucocorticoid influence on growth of vascular wall cells in culture

Primary mass cultures and cloned strains of bovine aortic endothelial and smooth muscle cells were investigated with respect to their growth responses to glucocorticoid hormones. The growth of primary endothelial cells was not influenced by glucocorticoid treatment in the absence of fibroblast growth factor (FGF) but was inhibited by about 30% in the presence of FGF; with cloned endothelial cells, glucocorticoids were also growth inhibitory only in the presence of FGF. In contrast, smooth muscle cell growth was inhibited 30%-70% by glucocorticoid treatment in both primary cultures and in the cloned strains in the absence of FGF, and this inhibition was totally abolished by the addition of FGF for both cultures. The corticosteroid influences on cell growth were glucocorticoid specific, concentration dependent, and were observed to be independent of the serum concentration. The results indicate that glucocorticoid hormones have direct and pronounced growth inhibiting effects on aortic smooth muscle cells but only minimal effects on endothelial cells when these components of the vascular wall are analyzed under identical conditions in vitro.     

Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells

HeLa cells grown in chemically defined medium lacking glucocorticoids synthesize metallothioneins, low molecular-weight heavy-metal binding proteins. Dexamethasone and hydrocortisone increase the rate of metal-lothionein synthesis five- to ten-fold. Maximal induction is achieved with 10^–8M dexamethasone and 10^–7M hydrocortisone. Half-maximal induction is achieved at 5 ± 10^–9M dexamethasone and 5 ± 10^–8M hydrocortisone. Although carried for many generations in the absence of any glucocorticoids, HeLa cells (clone S) contain 25,000 specific ³H-dexamethasone receptors that translocate into the nucleus after one hour of incubation. ³H-dexamethasone binds to a single class of receptors with an apparent Kd = 18.8 nM. A variety of steroids can be classified into three classes, based on their effect on metallothionein synthesis: (a) full agonists (optimal inducers), (b) intermediate effectors which have either partial agonist or antagonist activities, and (c) inactive steroids. There is a correlation between the effects on metallothionein synthesis of different steroids and their ability to compete with ³H-dexamethasone binding. We conclude that metallothionein is induced in HeLa cells by a glucocorticoid receptor mediated mechanism.     

Effect of dexamethasone,ammonium lons,and serum-deprivation on intracellular proteolysis in cultured muscle cells

Intracellular proteolysis was measured in primary cultures of newborn rat skeletal (gastrocnemius) and cardiac muscle cells by release to the medium of trichloroacetic acid-soluble label from cells grown in the presence of ¹⁴C-labeled phenylalanine. Exposure of the cultured cells to 10^?7M dexamethasone for 5 days starting at day 0 of culture resulted in an enhancement of proteolysis in skeletal muscle but not in cardiac muscle cells. Dexamethasone did not affect cell viability measured by release of label from cells preloaded with Na₂ ⁵¹CrO₄, release of creatine phosphokinase, and release of lactic dehydrogenase into the culture medium. Incorporation of ³H-thymidine into the cells increased during the first 3 to 4 days of culture and subsequently decreased, indicating that cell proliferation ceases at that time. When the exposure to dexamethasone was started on day 4 of culture, i.e., at a postmitotic stage, and continued for 4 days, proteolysis was again found to increase in skeletal but not cardiac cells, thereby suggesting that the response to the hormone is independent of the proliferative state of the culture. Ammonium chloride at a concentration of 10 mM produced a 50% reduction of the basal proteolysis in cultures of skeletal muscle cells and did not affect proteolysis in cardiac muscle cells. Exposure to ammonium chloride did not prevent the dexamethasone-induced increase of proteolysis in skeletal muscle cells. Serum-deprivation induced enhanced proteolysis which was not affected by NH₄Cl in both cell types. It is concluded that the differential responses of the two cultures to dexamethasone reflects the sparing of heart proteins and concomitant wasting of skeletal muscle proteins by glucocorticoid hormones in vivo, and that the enhancement of proteolysis by the glucocorticoid hormone or by serum-deprivation is not sensitive to the lysosomotropic agent NH₄Cl. Thus, while a lysosomal-autophagic enzyme system is responsible for almost half of the basal proteolysis, the accelerated proteolysis occurs via ammonium chloride-insensitive enzymes.