The distribution of centrosomes in endothelial cells of non-wounded and wounded aortic organ cultures

Summary The distribution of centrosomes in porcine vascular endothelial cells of the thoracic aorta maintained in organ culture was determined in en face preparations using immunofluorescence. Rectangular pieces of aorta that had the distal half (with respect to the heart) of their endothelial surface gently denuded with a scalpel blade and pieces with intact endothelium were cultured for up to 96 h. At time 0, centrosomes were found to be preferentially oriented toward the heart, both in the cells of intact monolayers and in cells at the wound edge. This distribution was maintained in the intact monolayers for at least 24 h, but by 72 h the number of centrosomes in the center of the cells exceeded the number oriented toward the heart as the cells changed from a fusiform to a polygonal shape. The centrosomes of most endothelial cells at the wound edge began to redistribute themselves within the first 24 h in culture, moving from a position toward the heart to a position either in the center of the cell or away from the heart. By 72 h, the majority of centrosomes in endothelial cells at the wound edge were oriented away from the heart toward the denuded region. It is concluded that the centrosomes in the endothelial cells maintained in organ culture respond to injury in a manner similar to those grown in monolayer cell culture except that the reorientation of centrosomes occurs more slowly.     

Culture of intramural cardiac ganglia of the newborn guinea-pig

The ultrastructure of cultured intrinsic neurones and SIF (small intensely fluorescent) cells dissociated from the atria and interatrial septum of newborn guinea-pig heart has been studied for the first time and compared with these cells in situ. Mononucleate and binucleate neuronal somata and their processes were observed in the culture preparation; their ultrastructure was similar to that of neurones in intracardiac ganglia observed in situ. The number of neurites associated with neuronal cell bodies increased after the first week in culture. A subpopulation of intracardiac neurones showed abnormalities in culture, comparable to the changes previously described in neurones of the monkey heart after unilateral vagotomy in situ. Small granule-containing cells were observed in culture, corresponding to those described in the heart in situ. One type of large process in the culture preparation containing densely packed mitochondria has not been seen in situ, suggesting that changes in cell ultrastructure due to the conditions of culture cannot be discounted. However, the ultrastructure of the cultured cells was, for the most part, consistent with that of the same cell type in situ, indicating that the culture preparation may be a useful model for investigation of the roles and interactions of intramural neurones in the heart, which are inaccessible for such studies in situ.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: III. The effect of cells extracted from rat cardiac allografts upon beating heart cell cultures

Cells extracted from rat cardiac allografts were able to bring about cessation of beating of heart cell culture monolayers nonspecifically. Nonadherent populations, depleted of macrophages, were consistently less potent than unseparated cells in this assay. Cells extracted from isografts were totally ineffective. Allogeneically stimulated peritoneal cells were also nonspecifically active. Again, nonadherent cells were less efficient than unseparated cells at stopping heart cell monolayers from beating, while adherent cells, enriched for macrophages, were more efficient. Activated bone marrow culture macrophages syngeneic or allogeneic to the heart cultures were also highly potent in beating heart cell assays. Thus in all cases the predominant effector cell type was adherent and nonspecific in its action and therefore presumably a macrophage. Supernatants from wells in which no beating cells remained following incubation with each type of effector population tested were transferred undiluted to fresh wells. In all cases there was no effect at all upon the beating of heart cell monolayers. Antirat heart antiserum plus complement was able to bring about the cessation of beating of heart culture monolayers at a dilution of 1:64. Alloantibody plus complement did not bring about cessation of beating at any dilution, although nonmyocardial cells were killed. The possibility that macrophages are the chief effector cell type in a DTH-like mechanism for cardiac allograft rejection is discussed.     

Oxygen consumption of mammalian myocardial cells in culture: measurements in beating cells attached to the substrate of the culture dish

A Lucite attachment which permitted the measurement of oxygen consumption in cells in culture without manipulating the cells was constructed. The attachment fit over commercially available dishes for cell culture and had an oxygen electrode built into it. Oxygen uptake of cells in culture was thus measured. Cells were attached to the substrate of the culture dish during the measurements and could be observed in an inverted phase microscope. Cells did not show any morphological changes, e.g., cell shapes or beating rate in case of myocardial cells, before and after the measurements of oxygen consumption. Using this method the rate of oxygen consumption was determined in rat myocardial and heart non-muscle cells in culture and also in HeLa and L6 cell lines. Myocardial cells in culture had an approximately four times higher rate of oxygen uptake compared with heart non-muscle, HeLa, and L6 cells. The oxygen uptake of beating myocardial cells was higher by about 50% compared with quiescent myocardial cells.     

Morphological transformation, autonomous proliferation and colony formation by chicken heart mesenchymal cells infected with avian sarcoma, erythroblastosis and myelocytomatosis viruses

Normal chicken heart mesenchymal cells at low density in monolayer culture in plasma-containing medium have a polygonal shape and are proliferatively quiescent. The combination of epidermal growth factor and insulin at hyperphysiological concentration, an insulin-like growth factor surrogate, causes these cells to assume a fusiform shape and to increase 40-fold in number during four days of incubation. These mitogenic hormones do not, however, induce normal chicken heart mesenchymal cells to form colonies in agarose suspension culture. Chicken heart mesenchymal cells infected with the Schmidt-Ruppin or Prague-A strains of Rous sarcoma virus or with the Fujinami or Y73 avian sarcoma viruses assume spindle and round shapes, increase 50-100 fold in number during four days of monolayer culture in the absence of mitogenic hormones and form macroscopic colonies during 3-4 days of agarose suspension culture. The autonomous (mitogenic hormone-independent) proliferation, in monolayer culture, of cells infected with temperature-sensitive transformation mutants of Rous sarcoma virus (tsNY68, tsNY72, tsLA24, tsLA29) is temperature-sensitive. Chicken heart mesenchymal cells infected with avian erythroblastosis virus assume spindle shapes and proliferate in monolayer culture at a rate comparable to that of sarcoma virus-infected cells but do not, however, form colonies in agarose suspension culture. Cells infected with the myelocytomatosis virus MC29 assume stellate shapes and increase 18-fold in number during four days of monolayer culture. Cells infected with the myelocytomatosis virus MH2 assume fusiform shapes and increase fourfold in number during four days of monolayer culture. Neither MC29 nor MH2 renders chicken heart mesenchymal cells capable of colony formation in agarose suspension culture. Infection with avian leukosis viruses (RAV-1, RAV-2, RPL-42) or with transformation-defective mutants of Rous sarcoma virus (tdNY105, 107, 109) does not affect the morphology or proliferative behavior of chicken heart mesenchymal cells. Monolayer culture of chicken heart mesenchymal cells in plasma-containing medium appears, therefore, to define the ability of onc genes of acute transforming avian retroviruses to induce autonomous (mitogenic hormone-independent) cell proliferation, the essential characteristic of neoplasia. The differences in transformed morphology and rates of autonomous proliferation between cells infected with different acute transforming retroviruses probably reflects differences in the modes of action of the transforming proteins encoded by the onc genes of the respective viruses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fatty acid and glucose oxidation by cultured rat heart cells

Monolayer cultures of fetal rat myocardial cells can be utilized to examine substrate preferences and interactions. The specific activity of glucose oxidation by myocardial cell cultures was high in sparse cultures but decreased with increased cell density. In contrast, palmitate oxidation was independent of initial cell density. Palmitate inhibited glucose oxidation by 50% in rat heart cultures. Glucose had only a slight sparing effect on palmitate oxidation. This suggests that fetal and newborn rat myocardial cells in culture preferentially oxidize palmitate similar to adult heart. The sparing effect of palmitate on glucose oxidation is accounted for by inhibition of the glycolytic-aerobic pathway and not by inhibition of the pentose phosphate pathway. Data on oxidation of ¹⁴C-pyruvate specifically labelled suggest that palmitate or a product of its oxidation such as acetyl-CoA may be acting directly to inhibit the pyruvate dehydrogenase complex. Palmitate oxidation per mg of cell protein was constant from 15 days gestational age to 2 days postnatal age. The observed differences between cultured cells and the intact heart may relate to decreased aerobic metabolism in monolayer cell culture and suggest that the increase in fatty acid oxidation observed in vivo is controlled by the oxygen environment of the cell. These studies show that heart cells in monolayer culture can be utilized to obtain metabolic information similar to an adult organ perfusion model.     

Modification by islet-activating protein of receptor-mediated regulation of cyclic AMP accumulation in isolated rat heart cells

Free cells isolated from adult rat heart by the collagenase method were maintained in culture up to 21 h with or without an islet-activating protein (IAP) that had been purified from the culture medium of Bordetella pertussis. Short-term stimulation of beta-adrenergic or glucagon receptors in these cultured cells caused more accumulation of cAMP in cells precultured with IAP (IAP-treated) than in nontreated cells, although there was no significant difference in the baseline (non-stimulated) content of cAMP between these cells. Stimulation of muscarinic cholinergic or adenosine R-site receptors caused a marked inhibition of cAMP accumulation in nontreated cells in either the presence or absence of a beta-agonist (or glucagon); no such inhibition was essentially observed in IAP-treated cells. These actions of IAP developed gradually and were dose-dependent with the half-maximal concentration of approximately 80 ng/ml in culture. It is concluded that IAP may exert its unique influence on the heart cell membrane causing profound modification of the coupling mechanism involved in the receptor-mediated activation or inhibition of adenylate cyclase. This action of IAP differs clearly from that of cholera toxin which activates adenylate cyclase rather independently of the receptor functions in heart cells.     

CHANGES IN GLUCOSE OXIDATION DURING GROWTH OF EMBRYONIC HEART CELLS IN CULTURE

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO₂. This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in ¹⁴CO₂ production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.     

Isolation,long-term culture,and ultrastructural characterization of adult cardiomyopathic cardiac muscle cells

Summary A long-term cell culture system for adult cardiomyopathic hamster cardiac muscle cells has been established. The diseased and control hearts were dissociated into single cell suspension with the modifications of our previous technique using collagenase and hyaluronidase as applied to the dissociation of the adult rat heart. The postperfusion of the diseased heart with Krebs-Ringer phosphate buffer and bovine serum albumin was very helpful in obtaining greater yield of viable diseased muscle cells; the cells were cultured for 4 wk. Approximately 60% of the myocytes from the diseased heart and 85% of the myocytes from the normal heart attached to the substrates and survived throughout the culture period. Approximately 60 to 70% of the cardiac myocytes from the diseased and control hearts were bi- or multinucleated; 30% of the diseased and 80% of the normal myocytes showed rhythmic contractility. Electron microscopy revealed the presence of two kinds of cardiac muscle cells in the diseased cell culture on the basis of their myofibril content: one with scanty myofibrils and another with abundant myofibrils. Myocytes with sparse myofibrils showed certain characteristic features that included autophagic vacuoles, amorphous matrix of fine filamentous texture, scattered strips of myofibrils, and abnormal organization of the Z-line. Cardiac muscle cells with abundant myofibrillar content contained unorganized myofibrils in certain sarcomeres. These studies demonstrate the feasibility of maintaining diseased cardiac muscle cells from adult cardiomyopathic hamsters for at least 4 wk in monolayer culture. This study was supported by a grant from the American Heart Association of Michigan, National Institutes of Health grant HL-25482, and by an Oakland University Biomedical Research Support Grant.     

Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart

Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in na?ve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.     

Culture of insect cells contracting spontaneously; research moving toward an environmentally robust hybrid robotic system

Here we propose an environmentally robust hybrid (biotic-abiotic) robotic system that uses insect heart cells. Our group has already presented a hybrid actuator using rat heart muscle cells, but it is difficult to keep rat heart muscle cells contracting spontaneously without maintaining the culture conditions carefully. Insect cells, by contrast, are robust over a range of culture conditions (temperature, osmotic pressure and pH) compared to mammalian cells. Therefore, a hybrid robotic system using not mammalian cells but insect cells can be driven without precise environmental control. As a first step toward the realization of this robotic system, the larvae of two lepidopteran species, Bombyx mori (BM) and Thysanoplusia intermixta (TI) were excised and the culture conditions of their dorsal vessel (insect heart) cells were examined. As a result, spontaneously contracting TI cells derived from the dorsal vessel were obtained. The contraction of TI cells started on the 7th day and continued for more than 18 days. Spontaneously contracting BM cells were not obtained in this study. These experimental results suggest the possibility of constructing an environmentally robust hybrid robotic system with living cells in the near future.     

Technical delivery of myogenic cells through an endocardial injection catheter for myocardial cell implantation

BACKGROUND: The next clinical frontier in the therapeutics of ischemic heart disease may involve the development and delivery of specific molecules and cells into the myocardium. The aim of the present study was to evaluate the efficiency and safety of the MyoStar injection catheter (Biosense-Webster Inc.) that has recently been developed to deliver molecules and cells to the myocardium. The 8 Fr (110 cm length) catheter comprises a navigation sensor with a 27 gauge needle at the distal tip. METHODS: Mouse myogenic cells (C2) were delivered to a tissue culture dish through different modalities: a standard laboratory pipette, a syringe needle (27 gauge) and the injection catheter. The cells were counted and monitored for growth and differentiation in the tissue culture immediately after delivery and two, three and six days later. Cells that were injected through a regular syringe needle or through the injection catheter demonstrated the same capacity to proliferate in tissue culture up to six days. RESULTS: The behavior of the cells in culture (fusion) was identical for the cells delivered to the tissue culture by a pipette or by the injection catheter. CONCLUSION: The results of the present study indicate that delivery of cells through the MyoStar injection catheter is a method with no significant loss or adverse effects to the cells along the path of the catheter. The catheter, which possesses both injection and navigation capabilities, can be used to deliver cell therapy to patients with ischemic heart disease.     

Culture of newt cells from different tissues and their expression of a regeneration-associated antigen

We have established culture conditions for cells from normal limb, early limb regenerate (blastema), heart, and liver of the newt Notophthalmus viridescens. Whereas heart and liver cells had a relatively short life in culture, limb cells have shown no sign of senescence over more than 1 year in culture. Cultured cells from all these tissues express to differing extents the regeneration-associated antigen 22/18. The antigen is intracellular and filamentous, and its expression appears to be regulated by culture density. Furthermore, 22/18 antigen is turned off in limb and blastemal cultures following differentiation into muscle, as also occurs in vivo.     

The expression of differentiation by chick embryo thyroid in cell culture. I. Functional and fine structural stability in mass and clonal culture

The stability of glandular epithelial cells has been investigated utilizing the techniques of cell culture. Embryonic chick thyroid was chosen as a representative cell type and methods were developed for the successful clonal culture of thyroid follicular cells. Thyroid cells were found to be morphologically and functionally stable while undergoing rapid division in both dense (monolayer) and dilute (clonal) cell culture. Differentiated features were retained for a minimum of 32 days of primary clonal culture (approximately 17 generations under clonal conditions). During the culture period, the cells retained their epithelial morphology, retained their cytoplasmic rough endoplasmic reticulum, and continued to produce chromatographically detectable thyroid hormones. Hormone production in culture was a specific thyroid characteristic since control cultures of embryonic heart and liver did not contain the hormones.     

Lipoprotein lipase in cultured heart cells: characteristics and cellular location.

Lipase activity extracted from cultured neonatal rat heart cells was characterized and identified as lipoprotein lipase. Enzyme activity was stimulated by human apoC-II and rat serum; serum stimulation was prevented by human apoC-I and by apoC-II. Lipolysis was maximal at pH 8.0 and was inhibited by protamine sulfate, NaCl, and high concentrations of heparin. About 50% of heart cell lipase activity applied to heparin-Sepharose bound to the gel and was eluted with a NaCl gradient. A peak of lipase activity was observed at 0.84 M NaCl. Neonatal rat heart cells in culture are a mixture of muscle and non-muscle cells. To determine the cellular location of the lipoprotein lipase, enzyme activity and muscle cell content of the cultures were determined. Myosin ATPase was used as an index of muscle cell content since ATPase specific activity correlated (r = +0.97) with muscle cell content determined immunofluorescently. When muscle cell content of cultures was decreased or increased by differential plating, lipase specific activity was constant. Moreover, lipase specific activity was constant during culture growth despite a decrease in muscle cell content. It was concluded that lipoprotein lipase activity of cultured heart cells is not associated solely with either muscle or non-muslce cells.     

Neurite outgrowth induced by the substrate associated material from nonneuronal cells

Cultured embryonic heart cells release a powerful inducer of neurite outgrowth into the surrounding medium. The present report demonstrates that these cells also deposit material which induces neurite outgrowth directly onto their culture substratum. Thus, embryonic heart cells condition both the culture medium and the culture substratum with respect to neurite outgrowth. Conditioned substrata were prepared by incubating heart cell monolayers in EDTA until the cells released from the substratum and were discarded. When dissociated neurons from ciliary or sympathetic chain ganglia were plated in fresh medium onto a conditioned substratum, neurite outgrowth was initiated in 80–95% of the neurons within 60 min. The neurite-inducing activity is trypsin sensitive, but is not inactivated by antibodies to the cell attachment protein fibronectin, by the membrane-solubilizing detergent Triton X-100, or by the enzymes collagenase, RNase, or DNase. The factor in conditioned medium which also induces neurite outgrowth depends for its activity on attachment to an artificial polyornithine substratum, under which condition it appears to promote adhesion of neuronal filopodia to the substratum. Thus, neurite outgrowth in these two culture systems occurs only if the substratum is conditioned by the appropriate extracellular materials: conditioned either directly by the deposition of heart cell products or indirectly by the binding of a conditioned medium factor to the polyornithine substratum. These substratum-conditioning factors may be related to those components of the extracellular matrix which support neurite outgrowth in vivo.     

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland)

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies.     

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.     

Efficient production of celery embryos and plantlets released in culture of immobilized gel beads

Celery embryos and plantlets were found to be selectively released in a culture of immobilized Ca-alginate gel beads in which celery callus was entrapped under regeneration conditions. We studied the feasibility of use of this process for celery embryogenesis in an artificial seed system. The cells released from the gel beads were larger than those obtained in suspension culture. The optimal concentration of alginate gel for embryo and plantlet production was 2% for the immobilized cell culture. Considering the maintenance of the gel bead structure and detrimental effect of CaCl₂ on plantlet development, 5 mM CaCl₂ supplementation gave the best result in terms of the number of heart and torpedo embryos and plantlets. The ratio of the number of heart embryos, torpedo embryos and plantlets to total number of cells in the immobilized cell culture was higher than that in the suspension culture. Repeated batch culture with 5 mM CaCl₂ provided long-term (more than 154 d) embryo and plantlet production without gel beads disruption. Productivity of plantlets in the immobilized cell culture with 5 mM CaCl₂ was 2.2-fold as high as that in the suspension culture.     

Biosynthesis of the Ca2+- and Mg2+-dependent adenosine triphosphatase of sarcoplasmic reticulum in cell cultures of embryonic chick heart.

The biosynthesis of the Ca2+- and Mg2+-dependent adenosine triphosphatase of sarcoplasmic reticulum was studied in cell cultures of embryonic chick heart. Rates of synthesis were estimated from the incorporation of tritium-labeled leucine into the ATPase. Newly synthesized ATPase was isolated from cells by immunoprecipitation. Radioactive leucine incorporation into the ATPase was determined by gel electrophoresis of the immunoprecipitates and counting of gel slices containing the ATPase band. Accumulation of the ATPase was estimated from the concentration of Ca2+ and Mg2+-dependent, hydroxylamine-sensitive phosphoprotein in the whole cell membrane fraction of cultured cells. Embryonic heart cells cultured in a medium which permitted cell proliferation showed approximately linearly increasing rates of ATPase synthesis and accumulation/culture plate as the cells proliferated. When cells were cultured in a serum-free medium, cell proliferation was inhibited and there was no sustained increase in the rate of ATPase synthesis or accumulation. Inclusion of isoproterenol or dibutyryl cyclic AMP at concentrations of 10 microM up to 1 mM in serum-free culture medium failed to stimulate significantly ATPase synthesis.