The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

The effects of mastoparan on the carrot cell plasma membrane polyphosphoinositide phospholipase C.

When [3H]inositol-labeled carrot (Daucus carota L.) cells were treated with 10 or 25 microM wasp venom peptide mastoparan or the active analog Mas-7 there was a rapid loss of more than 70% of [3H]phosphatidylinositol-4-monophosphate (PIP) and [3H]phosphatidylinositol-4,5-bisphosphate (PIP2) and a 3- and 4-fold increase in [3H]inositol-1,4-P2 and [3H]inositol-1,4,5-P3, respectively. The identity of [3H]inositol-1,4,5-P3 was confirmed by phosphorylation with inositol-1,4,5-P3 3-kinase and co-migration with inositol-1,3,4,5-P4. The changes in phosphoinositides were evident within 1 min. The loss of [3H]PIP was evident only when cells were treated with the higher concentrations (10 and 25 microM) of mastoparan or Mas-7. At 1 microM Mas-7, [3H]PIP increased. The inactive mastoparan analog Mas-17 had little or no effect on [3H]PIP or [3H]PIP2 hydrolysis in vivo. Neomycin (100 microM) inhibited the uptake of Mas-7 and thereby inhibited the Mas-7-stimulated hydrolysis of [3H]PIP and [3H]PIP2. Plasma membranes isolated from mastoparan-treated cells had increased PIP-phospholipase C (PLC) activity. However, when Mas-7 was added to isolated plasma membranes from control cells, it had no effect on PIP-PLC activity at low concentrations and inhibited PIP-PLC at concentrations greater than 10 microM. In addition, guanosine-5'-O-(3-thiotriphosphate) had no effect on the PIP-PLC activity when added to plasma membranes isolated from either the Mas-7-treated or control cells. The fact that Mas-7 did not stimulate PIP-PLC activity in vitro indicated that the Mas-7-induced increase in PIP-PLC in vivo required a factor that was lost from the membrane during isolation.     

Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C

The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on l-leucyl-β-naphthylamidase, alkaline phosphodiesterase I and Ca²⁺- or Mg²⁺-ATPase, but substantial proportions of the alkaline phosphatase and 5′-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not excluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.     

Regulation of goblet cell differentiation by calcium in embryonic chick intestine

The potential role of calcium as a regulator of goblet cell differentiation was tested by culturing duodenal explants from 14-day chicken embryos in media containing a range of calcium concentrations. Extracellular calcium in vitro approximated the range of plasma calcium concentration that was found to rise by 16% during the third week of embryonic development. As ionic calcium was increased from 0.9 to 2.0 mM in 48-h cultures, the number of goblet cells per 100 ridges increased progressively from 29 +/- 2.2 to 158 +/- 6.9 while attaining a distribution on the previllous ridges similar to that in ovo. The rate of goblet cell differentiation in vitro exceeded that in ovo when extracellular calcium was increased to 1.1 mM and was maximal at 1.6-2.0 mM calcium. Neither the growth of previllous ridges nor gross morphology of the tissue was altered as extracellular calcium was increased. Goblet cell differentiation in 1.3 mM calcium was stimulated by 0.1 microM calcium ionophore and inhibited by the calmodulin antagonist trifluoperazine, indicating an intracellular site for calcium action. These results indicate that calcium plays a primary role in regulating goblet cell differentiation in embryonic intestine and suggest that rising levels of plasma calcium influence the rate of differentiation in ovo.     

Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis

The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [(14)C]oleate were treated with PLC from Clostridium perfringens for 6 h, [(14)C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of (14)C label was even greater when the cells were simultaneously exposed to PLC and 100 microM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [(3)H]glycerol, a subsequent increase in [(3)H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 microM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG.These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.-Igal, R. A., J. M. Caviglia, I. N. T. de Gómez Dumm, and R. A. Coleman. Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis. J. Lipid Res. 2001. 42: 88;-95.     

Specific release of plasma membrane enzymes by a phosphatidylinositol-specific phospholipase C.

The release of plasma membrane ecto-enzymes by a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was investigated. There was no effect on L-leucyl-beta-naphthylamidase, alkaline phosphodeisterase I and Ca2+- or MG2+-ATPase, but substantial proportions of the alkaline phosphatase and 5-nucleotidase were released. There was no simultaneous release of phospholipid and the solubilized enzymes were not exluded from Sepharose 6-B. It was therefore concluded that release was not a secondary consequence of membrane vesiculation but occurred as a result of the disruption of specific interactions involving the phosphatidylinositol molecule.     

The ultrastructure of polygonal networks in chick embryonic cells in vitro

Polygonal networks in cultured chick endoderm cells are ordered arrays of actin microfilaments situated just beneath the dorsal cell surface. Each strut is formed from a bundle of microfilaments and 5-7 bundles intersect at each node. Dense bodies are seen in nodes and some struts. At its periphery the network is attached to the substrate at the termini of long radial struts. Most of the network is resistant to detergent extraction. Sliding microfilaments can explain the observed behaviour of networks in live cells.     

Changes in membrane properties of chick embryonic hearts during development

The electrophysiological properties of embryonic chick hearts (ventricles) change during development; the largest changes occur between days 2 and 8. Resting potential (E_m) and peak overshoot potential (+E _max) increase, respectively, from -35 mv and +11 mv at day 2 to -70 mv and +28 mv at days 12–21. Action potential duration does not change significantly. Maximum rate of rise of the action potential (+V _max) increases from about 20 v/sec at days 2–3 to 150 v/sec at days 18–21; + V _max of young cells is not greatly increased by applied hyperpolarizing current pulses. In resting E_m vs. log [K⁺]_o curves, the slope at high K⁺ is lower in young hearts (e.g. 30 mv/decade) than the 50–60 mv/decade obtained in old hearts, but the extrapolated [K⁺]_i values (125–140 mM) are almost as high. Input resistance is much higher in young hearts (13 MΩ at day 2 vs. 4.5 MΩ at days 8–21), suggesting that the membrane resistivity (R_m) is higher. The ratio of permeabilities, P _Na/P _K, is high (about 0.2) in young hearts, due to a low P _K, and decreases during ontogeny (to about 0.05). The low K⁺ conductance (g _K) in young hearts accounts for the greater incidence of hyperpolarizing afterpotentials and pacemaker potentials, the lower sensitivity (with respect to loss of excitability) to elevation of [K⁺]_o, and the higher chronaxie. Acetylcholine does not increase g _K of young or old ventricular cells. The increase in (Na⁺, K⁺)-adenosine triphosphatase (ATPase) activity during development tends to compensate for the increase in g _K. +E _max and + V _max are dependent on [Na⁺]_o in both young and old hearts. However, the Na⁺ channels in young hearts (2–4 days) are slow, tetrodotoxin (TTX)-insensitive, and activated-inactivated at lower E_m. In contrast, the Na⁺ channels of cells in older hearts (> 8 days) are fast and TTX-sensitive, but they revert back to slow channels when placed in culture.     

Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain

An immunohistochemical survey was carried out on frozen sections of the early embryonic chick brain between 1 and 6 days of incubation, with antisera to the three neurofilament proteins (NF-L, NF-M, NF-H). Large numbers of replicating neuroepithelial cells were found to express one of these proteins, NF-M, generations before the existence of any postmitotic neuroblasts (Days 1-2 1/2 of incubation). NF-L and NF-H could not be detected. Not all primordial brain regions contained NF-M-positive cells, but in those that did, every cell was positive. These regions included the dorsal forebrain, optic vesicles, and dorsal hindbrain, but not the dorsal midbrain. All cells in all regions of the cephalic neural tube contained vimentin, whether or not they also contained NF-M. This NF-M expression was transient in the sense that later generations of these NF-M-positive neuroepithelial cells became NF-M negative, before finally giving rise to some descendents that ultimately express all three NF proteins. This transient NF-M expression was found in certain other cells of early embryos, including cardiac myoblasts. The identity of the component in these early neural and nonneural tissues, that bound the antibody, was demonstrated to be identical to adult brain NF-M by one- and two-dimensional immunoblots. These findings demonstrate an unusual kind of biochemical heterogeneity among neuroepithelial cells, and they are relevant to considerations regarding lineage analysis and lineage "markers" in the vertebrate central nervous system.     

Hemopoietic colonies on the chorioallantoic membrane of the chick embryo: Induction by embryonic,adherent, non-hemopoietic spleen cells

Granulocytic and erythrocytic colonies developed on the chick embryo chorioallantoic membrane (CAM) following the inoculation of chick embryo spleen cells. Dose response and kinetic experiments showed that the colonies were derived from cell aggregates present in the inoculum. Dissociation and reaggregation studies of the CAM colony-inducing cells (CAM-CIC) indicated that these cells must be present as aggregates in order to form colonies. Results from the morphology and cell marker experiments suggested that the colony-inducing aggregates (CAM-CIA) attract and support the differentiation of primitive host hemopoietic cells. The physical characteristics of the CAM-CIC, which are different from those of the hemopoietic progenitor cells, indicated that they represent a stromal cell population of the chick embryo spleen. Further evidence supporting this notion was provided by the radiation studies which showed that the colony-inducing ability of the CAM-CIC is radioresistant. The above characteristics of the CAM-CIC strongly suggest that they represent the stromal cells of the chick embryo spleen which influence hemopoiesis.     

Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.

During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.     

The in vitro cell fusion of embryonic chick muscle without DNA synthesis

A system has been developed for the in vitro development of chick skeletal muscle monolayers, in which a burst of synchronous fusion occurs, such that some 40% of the spindle-shaped cells fuse in a 10-hr period. Cells inhibited from synthesizing DNA by ara-C do fuse, but at a later time than the normal burst. If ara-C is added to cultures 6 hr or more before the normal fusion time, fusion is delayed, but no delay results when the drug is added after this time. A medium change will delay the fusion if done 4 hr or more before fusion, but gives no delay if done later. Cells grown in conditioned medium fuse some 10 hr earlier than controls, even in the presence of ara-C, as do cultures prepared at higher than normal cell densities. The data suggest that muscle cell fusion is independent of DNA synthesis in vitro, but depends upon a modification of the culture medium to a sufficient degree required for initiating the synthetic program for fusion.     

The electrical activity of embryonic chick heart cells isolated in tissue culture singly or in interconnected cell sheets

Embryonic chick heart cells were cultured on a plastic surface in sparse sheets of 2–50 cells mutually in contact, or isolated as single cells. Conditions are described which permitted conjoint cells to be impaled with recording microelectrodes with 75 % success, and isolated single cells with 8 % success. It is proposed that cells in electrical contact with neighbors are protected from irreversible damage by the penetrating electrode, by a flow of ions or other substances from connected cells across low-impedance intercellular junctions. Action potentials recorded from conjoint and isolated single cells were similar in form and amplitude. The height or shape of the action potential thus appears not to depend upon spatial relationships of one cell to another. As the external potassium concentration was increased from 1.3 mM to 6 mM, cells became hyperpolarized while the afterhyperpolarization was reduced. At higher potassium levels, the afterhyperpolarization disappeared, the slope of the slow diastolic depolarization decreased, and resting potential fell along a linear curve with a slope of 61 mv per 10-fold increase in potassium. In pacemaker cells the diastolic depolarization consists of two phases: (a) recovery from the afterpotential of the previous action potential and (b) the pacemaker potential. These phases are separated by a point of inflection, and represent manifestations of different mechanisms. Evidence is presented that it is the point of inflection (PBA) rather than the point of maximal diastolic potential, that should be taken as the resting potential.     

Okadaic acid blocks membrane fusion of chick embryonic myoblasts in culture

Okadaic acid was found to block membrane fusion of chick embryonic myoblasts in culture. It also induced morphological change of the cells from bipolar to spherical shape. These effects were dose-dependent, and could be reversed upon removal of the drug from the culture medium. It showed, however, no effect on the induction of muscle specific proteins including tropomyosin and creatine kinase. When okadaic acid was treated to the cell lysates, the phosphorylation state of many proteins significantly increased. These results suggest that the inhibition of myoblast fusion by okadaic acid may be mediated by the increase in the phosphorylation of certain, unknown protein(s) that regulate the fusion process.