Maercklein, Peter B.

ABSTRACT The topography of plasma membrane intramembranous particles in contact-inhibited and transformed cells is a topic of current controversy. We have reported that particles are aggregated in 3T3 cells and that they are randomly distributed in SV3T3 cells. Other investigators have, however, proposed that these differences result from the effect of cryoprotectants and are blocked by aldehyde fixation. The experiments described in this paper re-examine PMP topography in large samples of 3T3 and SV3T3 cells prepared by a variety of preparative methods. The results confirm that 3T3 cells contain aggregated intramembranous particles and that native SV3T3 cells do not, regardless of whether cells are prepared in glycerol, sucrose, tissue culture medium or following prefixation in paraformaldehyde. Data presented in this paper also show that cytochalasin B induces dose -and time-dependent disaggregations of particles in contact-inhibited 3T3 cells. Particle disaggregation results from exposure of cells in situ for 1-3 h to 1-500 ng/ml cytochalasin B. Removal of the drug by washing with reincubation of cells in medium containing serum results in particle reaggregation within 1 to 3 h. These observations suggest that intramembranous particles in 3T3 cells are aggregated in the native state and that membrane-active drugs modulate their distribution by a mechanism possibly involving disruption of linkages between membrane components and the cytoskeleton.

ABSTRACT Plasma membrane vesicles are shed from monolayer cell cultures during incubation in low concentrations of formaldehyde and other sulphydryl blocking reagents. In both 3T3 and SV3T3 cells disulphide reducing agents, including dithiothreitol and mercaptoethanol, potentiate formaldehyde-induced vesiculation. Plasma membrane vesiculation is shown to be a temperature-dependent phenomenon which occurs optimally between 22 and 37 °C and at pH 7·0 to 7·5. Membrane shedding is an energy-dependent phenomenon, requiring monovalent and divalent cations and slightly hypertonic medium. Plasma membrane vesiculation is not affected by pretreatment of cells with inhibitors of protein synthesis, i.e. cycloheximide, nor by agents which disrupt the cytoskeleton.

ABSTRACT 3T3 and SV3T3 mouse embryo cells and a variety of other monolayer cell lines can be induced to form and shed plasma membrane vesicles by exposure to sulphydryl blocking agents including formaldehyde and N-ethyl malemide. Morphological studies show that multiple vesicles are formed and released from individual cells and that the vesicle membrane is continuous with the plasma membrane of the cell. Vesicles measure from 0 ·1 to 15 μm in diameter and are free of detectable contamination with cytoplasmic membranes and organelles. Vesicles also show a 10-fold enrichment in the plasma membrane marker enzyme 5’nucleotidase and are devoid of detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which are marker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles have a high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. They contain receptors for Con A and WGA, approximately 20 size class polypeptides and intra-membranous particles. These results suggest that vesicles are derived from and have the general characteristics of plasma membranes.

It currently remains controversial as to the intracellular mechanisms coupled to the inhibition of parathyroid hormone (PTH) secretion that are modulated by extracellular divalent cations. To study mechanisms responsible for regulation of PTH release by cations, we investigated the effect of Mg2+ on cytosolic Ca2+ levels ([Ca2+]i) and PTH secretion in single isolated bovine parathyroid cells. Addition of 9.0 mM Mg2+ evoked a spike of [Ca2+]i in approximately 80% of cells in the presence of extracellular Ca2+. Mg2+ decreased steady-state [Ca2+]i, which represented inhibition of influx of extracellular Ca2+ in 13-78% of cells. The percentage of cells that had a decline in steady-state [Ca2+]i after exposure to Mg2+ was dependent on the extracellular Ca2+ concentration. The effect of Mg2+ on intracellular Ca2+ response was dose dependent. Extracellular Mg2+ inhibited PTH secretion in cells that showed decline in steady-state [Ca2+]i, although cells that showed a spike after addition of Mg2+ secreted more PTH than cells that did not show a spike. The spike of [Ca2+]i and decline in steady-state [Ca2+]i that occur in response to extracellular Mg2+ may be caused independently by two distinct mechanisms that differentially regulate secretion of PTH.

Abstract Cell-cell interactions are important in the regulation of endocrine cell secretion. To investigate the possibility that cell communication may alter the regulation of parathyroid cell secretion, we utilized the reverse hemolytic plaque assay (RHPA) to measure parathyroid hormone (PTH) release from individual cells. Bovine parathyroid cells were dispersed and plated with protein A-conjugated erythrocytes at cell densities ranging from 0.9 to 36 X 102 cells/cm2 in 0.2 mM calcium. Cell populations were greater than 98% homogeneous as determined by immunocytochemistry and in situ hybridization for PTH mRNA. Plaques were developed and data analyzed for the amount of PTH per cell released (plaque area in μm2 X 104) and the determination of cell recruitment (% plaques formed). A positive correlation existed between parathyroid cell density and the amount of PTH released. As the distance between cells increased, the plaque area (amount of PTH released per cell) decreased (ranging from 1.0 X 1044 μm2 at 0.9 X 102 cells/cm2 versus 1.6 X 104 μm2 at 36 X 102 cells/cm2). The percentage of cells releasing PTH (recruitment) also decreased (16% at 0.9 X 102 cells/cm2 versus 47% at 36 X 102 cells/cm2). These data suggest that parathyroid cells in close proximity are stimulated to secrete more hormone than those at lesser densities. In addition, parathyroid cells are recruited to secrete PTH when plated at high density. Factor(s) released by the parathyroid cell may increase cell responsiveness and stimulate secretion in a paracrine fashion.

The potential of nonterminal cellular differentiation to stably repress the expression of the neoplastic phenotype of transformed cells is established. Nonterminal differentiation induces spontaneously transformed 3T3 T cells to revert to a nontransformed state and induces the revertant cell clones to become resistant to retransformation by UV irradiation or 4-nitroquinoline oxide treatment. Nonterminal differentiation also induces simian virus 40-transformed 3T3 T cells to repress expression of the large tumor antigen and to revert to a nontransformed state. Although the molecular mechanisms that mediate these and other forms of anticancer activity have not been definitively established, data are presented which suggest that differentiation-induced repression of large tumor antigen expression can be regulated at the level of transcription and/or RNA processing.