Biomedical subjects

D Bar-Sagi

Publications and source records attributed to D Bar-Sagi.

67 records · Page 4Linked to original sources

PubMed1989

G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2.

Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers.

Animals↗

PubMed1988

Localization of phospholipase A2 in normal and ras-transformed cells.

The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed.

Animals↗

PubMed1988

Stimulation of exocytotic degranulation by microinjection of the ras oncogene protein into rat mast cells.

To investigate the possible role of ras proteins in the secretory process, we have microinjected the proto oncogenic and oncogenic forms of the human H-ras protein into rat peritoneal mast cells. Mast cells are secretory cells which, upon appropriate stimulus, liberate histamine and other mediators of the acute inflammatory reaction by exocytotic degranulation. We report here that microinjection of the ras oncogene protein into mast cells induces exocytotic degranulation. In contrast, microinjection of similar amounts of the proto-oncogenic protein has little apparent effect on mast cells. Degranulation induced by injection of the ras oncogene protein occurs in the absence of an external stimulus and requires the presence of external calcium. The ultrastructural features of exocytotic degranulation in mast cells injected with the ras oncogene protein are similar to those seen when mast cells are activated by soluble ligands. Our results suggest that ras proteins may be involved, possibly as regulatory elements, in cellular functions that control exocytosis.

Animals↗

PubMed1987

Inhibition of cell surface ruffling and fluid-phase pinocytosis by microinjection of anti-ras antibodies into living cells.

Fibroblasts transformed by ras oncogenes display enhanced cell surface ruffling and fluid-phase pinocytotic activities. Microinjection of antibodies that specifically bind the ras proteins into these cells results in the inhibition of these two surface activities. The possible underlying biochemical basis of the influence of the ras proteins on membrane ruffling and pinocytosis and the potential relationship of these two biological activities to membrane signal transduction are discussed.

Animals↗

PubMed1987

Regulation of membrane turnover by ras proteins.

Because ras oncogenes mediate abnormal cellular growth, ras proteins have been presumed to play a role primarily in growth control. The biological function of ras proteins may, however, prove to be much more diverse: ras proteins may be involved in cellular functions that control endocytosis and/or exocytosis.

Cell Membrane↗

PubMed1986

Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins.

Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins.

Animals↗

PubMed1985

Negative modulation of sodium channels in cultured chick muscle cells by the channel activator batrachotoxin.

We have investigated the possibility that cellular control of membrane excitability involves feedback mechanisms in which the degree of activity of voltage-sensitive Na+ channels regulates the number of these channels. Using two independent assays, channel-mediated Na+ uptake and the specific binding of [3H] saxitoxin, we have studied the effects of pharmacological activation of Na+ channels with batrachotoxin (BTX) on the number and properties of these channels. Upon exposure of cultured muscle cells to BTX (1 microM), the number of surface Na+ channels decreases by approximately 75%, with a half-time of 3-6 h. This decrease is prevented by pharmacological blockade of these channels and does not reflect changes in the apparent affinities towards either BTX or saxitoxin. This reduction is reversible: a gradual increase in surface Na+ channels that is dependent on protein synthesis is observed upon removal of the activator. The BTX-induced decrease in Na+ channels is associated with an enhanced rate of disappearance of surface Na+ channels. These findings point to the existence of a down-regulation mechanism for the modulation of membrane excitability under conditions of elevated Na+ channel activity.

Animals↗

PubMed1985

Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation.

To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H-ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein.

Adrenal Gland Neoplasms↗

PubMed1983

Effect of tunicamycin, an inhibitor of protein glycosylation, on the biological properties of acetylcholine receptor in cultured muscle cells.

We have studied the effect of tunicamycin (TM), an antibiotic which inhibits the glycosylation of nascent proteins, on the properties of the acetylcholine receptor (AChR) at the surface of embryonic chick skeletal muscle cells. The use of two separate assays, specific binding of 125I-alpha-bungarotoxin and carbamylcholine-activated 22Na+ uptake, has allowed us to monitor the effects of impaired glycosylation on the metabolic and functional properties of AChR. A significant decrease in the amounts of surface AChR elaborated in the presence of TM is detected by both measurements. This decrease has been found to reflect an enhanced proteolytic degradation of the underglycosylated AChR. The underglycosylated AChR, expressed on the cell surface in the presence of TM, retains the capability of mediating agonist-activated ionic permeability changes, but displays quantitatively altered interactions with receptor ligands. We conclude that the carbohydrate moiety on AChR may play a role in determining the folding of newly synthesized polypeptides to form a conformation compatible with the metabolic properties and ligand interactions characteristic of glycosylated AChR.

Animals↗

PubMed1983

Tunicamycin inhibits the expression of surface Na+ channels in cultured muscle cells.

We have investigated the effect of tunicamycin (TM), an inhibitor of protein glycosylation, on surface Na+ channels in cultured chick skeletal muscle cells. The expression of Na+ channels, estimated by the measurement of batrachotoxin (BTX)-activated 22Na+ uptake, was found to be significantly diminished after exposure of muscle cells to TM. This effect is partially reversed by the protease inhibitor leupeptin and is associated with a markedly enhanced rate of disappearance of Na+ channels from the surface of TM-treated cells. Our findings suggest that protein glycosylation contributes to the metabolic stability of voltage-sensitive Na+ channels.

Animals↗

PubMed1983

Trifluoperazine, a calmodulin antagonist, inhibits muscle cell fusion.

We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1-naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion.

Animals↗

PubMed1983

Differential expression of sodium channel activities during the development of chick skeletal muscle cells in culture.

The expression of Na+ channels during differentiation of cultured embryonic chick skeletal muscle cells was investigated using saxitoxin (STX) and batrachotoxin (BTX), which previously have been shown to interact with distinct, separate receptor sites of the voltage-sensitive Na+ channel of excitable cells. In the present study, parallel measurements of binding of [3H]-STX (STX) and of BTX-activated 22Na+ uptake (Na influx) were made in order to establish the temporal relationship of the appearance of these two Na+ channel activities during myogenesis. Na influx was clearly measurable in 2-d cells; from day 3 to day 7 the maximum Na influx approximately doubled when measured with saturating BTX concentrations potentiated by Leiurus scorpion toxin, while the apparent affinity of BTX, measured without scorpion toxin, also increased. Saturable STX binding did not appear consistently until day 3; from then until day 7 the STX binding capacity increased about threefold, whereas the equilibrium dissociation constant (KD) decreased about fourfold. Although Na influx in cells of all ages was totally inhibited by STX or tetrodotoxin (TTX) at 10 microM, lower concentrations (2-50 nM) blocked the influx in 7-d cells much more effectively than that in 3-d cells, where half the flux was resistant to STX at 20-50 nM. Similar but smaller differences characterized the block by TTX. In addition, when protein synthesis is inhibited by cycloheximide, both Na influx and STX binding activities disappear more rapidly in 3-d than in 7-d cells, which shows that these functions are less stable metabolically in the younger cells.

Animals↗

PubMedDate not supplied

ras proteins: biological effects and biochemical targets (review).

Ras genes are an ubiquitous eukaryotic gene family. Since their discovery as the cellular homologues of the transforming genes of Harvey and Kirsten retroviruses, ras genes have been presumed to play a role in growth control, mainly because of their potential to induce uncontrolled cell proliferation. This notion is strongly supported by recent evidence indicating that ras mutations may be causative or closely linked to the onset of some types of human tumors. However, the mechanism of action of ras proteins in mammalian cells is poorly understood. Using the microinjection technique as a biological assay for ras proteins, it has been possible to address several important questions concerning cellular and biochemical aspects of ras function. When introduced into living cells by microinjection, purified ras proteins can induce cell proliferation, neuronal differentiation, oocyte maturation, and exocytotic degranulation. On the biochemical level, microinjection studies indicated that ras proteins can induce specific alterations in phospholipid metabolism.

Animals↗