Perturbing cell surface beta-(1,4)-galactosyltransferase on F9 embryonal carcinoma cells arrests cell growth and induces laminin synthesis.

Cell growth and differentiation are influenced by intercellular contact, suggesting that cell adhesion molecules may be instrumental in triggering these events. F9 embryonal carcinoma cells are an ideal system in which to examine the function of cell adhesion molecules in growth and differentiation, since the relevant cell adhesion molecules and differentiation markers are well defined. Intercellular adhesion in F9 cells is mediated by uvomorulin, or E-cadherin, and cell surface beta-(1,4)-galactosyltransferase. Since previous studies suggested that neither F9 cell growth nor differentiation is directly dependent on uvomorulin function, in this study we examined whether cell surface galactosyltransferase plays any role in F9 cell growth or differentiation. A variety of galactosyltransferase perturbants, including anti-galactosyltransferase antibodies, UDPgalactose, and the substrate modifier protein alpha-lactalbumin, inhibited the growth of F9 cells, whereas control reagents did not. To examine this in more detail, we analyzed the effects of perturbing surface galactosyltransferase on progression through the F9 cell cycle. Anti-galactosyltransferase IgG treatment inhibited ornithine decarboxylase activity and lengthened the F9 cell cycle during G1 and G2, the latter mimicking the effects of retinoic acid, a reagent known to prolong the F9 cell cycle and induce differentiation. In contrast, anti-uvomorulin antibodies had no effect on F9 cell growth, ornithine decarboxylase activity, or progression through the cell cycle. Furthermore, perturbation of surface galactosyltransferase adhesions in F9 cell aggregates induced precocious F9 cell differentiation, as assayed by increased laminin synthesis, whereas control reagents had no effect. Thus, perturbing surface galactosyltransferase adhesions in F9 cells both decreases growth and stimulates synthesis of laminin. These results imply that interactions between surface galactosyltransferase and its oligosaccharide ligand during cell adhesion may affect the normal growth-regulatory and differentiation-inducing signals, as is seen, in part, during treatment with retinoic acid.

Uvomorulin, LAMP-1, and laminin are substrates for cell surface beta-1,4-galactosyltransferase on F9 embryonal carcinoma cells: comparisons between wild-type and mutant 5.51 att- cells.

Intercellular adhesions in F9 embryonal carcinoma cells are primarily dependent upon two types of cell adhesion molecules: uvomorulin (Um or E-cadherin), which facilitates intercellular adhesion by homophilic binding, and cell surface beta-1,4-galactosyltransferase (GalTase), which binds terminal N-acetylglucosamine residues on consociate glycoprotein substrates on adjacent cell surfaces. The variant F9 cell line, 5.51 att-, undergoes initial cell aggregation, but fails to form the mature intercellular adhesions characteristic of wild-type F9 cells. The 5.51 att- cells show reduced Um expression; however, previous studies have shown that reduced levels of Um are not responsible for the att- mutant phenotype. Therefore, in this study we determined whether the att- mutant phenotype was the result of altered expression of surface GalTase or of its consociate glycoprotein substrates. Surface GalTase was found to be expressed normally on 5.51 att- cells. Furthermore, the initial intercellular adhesions characteristic of reaggregating 5.51 att- cells were dependent upon surface GalTase, since anti-GalTase antibodies inhibited 5.51 intercellular adhesion. Preliminary studies showed that the interaction of surface GalTase with its consociate glycoprotein substrates was abnormal on 5.51 att- cells. To define the biochemical basis for this observation, specific glycoprotein substrates for cell surface GalTase were identified and their expression and utilization were compared between wild-type and 5.51 att- cells. In wild-type cells, uvomorulin (Um), lysosome-associated membrane protein-1 (LAMP-1), and laminin (Lm) were shown to be substrates for cell surface GalTase, suggesting that they may participate in GalTase-specific adhesions. Moreover, the interaction between GalTase and these glycoproteins exhibited characteristic changes during retinoic acid-induced F9 cell differentiation. In contrast to that seen on wild-type cells, surface GalTase interaction with Um, LAMP-1, and Lm was atypical on 5.51 att- cells, resulting, in part, from increased synthesis of oligosaccharide substrates for surface GalTase, as determined by size exclusion and lectin affinity chromatography. N-Acetylglucosaminyltransferase activity was elevated in 5.51 att- cells and likely responsible for the increased expression of GalTase oligosaccharide substrates. These results suggest that mutant 5.51 att- cells are able to maintain residual intercellular adhesions because of increased expression of oligosaccharide substrates for cell surface GalTase. The impact of aberrant GalTase-specific adhesions on the ability of 5.51 att- cells to form mature intercellular adhesions and to differentiate in response to retinoic acid is discussed.

Cell surface and Golgi pools of beta-1,4-galactosyltransferase are differentially regulated during embryonal carcinoma cell differentiation.

beta-1,4-Galactosyltransferase (GalTase) has two functionally distinct subcellular distributions. In the Golgi apparatus, GalTase participates in the glycosylation of secretory and membrane-bound glycoproteins, whereas on the cell surface it mediates specific aspects of intercellular adhesion. For this study, a murine GalTase clone was obtained by screening a lambda gt10 cDNA library made from F9 embryonal carcinoma cells with a heterologous bovine GalTase cDNA probe. The murine GalTase cDNA probe was used in conjunction with assays of GalTase activity to investigate the expression and distribution of GalTase during differentiation of F9 stem cells into secretory endodermal epithelium. During the initial phase of F9 cell differentiation, GalTase mRNA levels remained relatively constant; however, as differentiation progressed, as assayed by expression of the differentiation-specific marker laminin B1, GalTase mRNA levels and enzyme activity rose dramatically. Furthermore, subcellular fractionation of these cells showed that the increased GalTase levels were specifically associated with the Golgi apparatus, whereas GalTase specific activity on the plasma membrane remained constant. These results show that levels of cell surface and Golgi GalTase change relative to one another during F9 cell differentiation and suggest that these functionally distinct pools of GalTase are independently and differentially regulated.

Adrenergic and serotonergic regulation of skeletal muscle metabolism in the rat: specificity of the serotonin- and isoproterenol-stimulable adenylyl cyclase in sarcolemma.

The specificity and kinetics of rat sarcolemmal serotonin- and isoproterenol-stimulable adenylyl cyclases were studied. The stimulation of adenylyl cyclase by serotonin was less than that by isoproterenol and required low concentrations of ATP (0.1 mM), 10 microM GTP, and free Mg ion concentrations of 10-20 microM. The isoproterenol-stimulated activity was readily detectable over a much wider range of MgCl2 (1.0-5.0 mM) and ATP (0.1-3.0 mM). (+/-)-Oxprenolol and l(-)-propranolol inhibited isoproterenol stimulation with KB values of 6.3 X 10(-9) M and 1.9 X 10(-9) M, respectively. Interference by (+/-)-cyproheptadine, (+/-)-methysergide and d(+)-propranolol was not strictly competitive. However, these were much more potent inhibitors of serotonin stimulation than were oxprenolol and l-propranolol. The concentrations of antagonists producing 50% inhibition (IC50) of the maximal adenylyl cyclase stimulation by serotonin were compared to the IC50 obtained with isoproterenol; the ratios were 0.02 for oxprenolol, 0.50 for l-propranolol, 295 for d-propranolol, 2438 for cyproheptadine, 578 for methysergide and greater than 125,000 for lysergic acid diethylamide (LSD). These data indicate that rat skeletal muscle adenylyl is stimulated by discrete serotonergic and adrenergic receptors, and that there may be distinct conditions for optional stimulation.

The effect of hemodialysis on levels of cyclic nucleotide-associated hormones in patients with chronic renal failure.

In order to determine the molecular basis for the loss of catecholamine responsiveness in skeletal muscle in chronic azotemia, plasma and serum levels of catecholamines and other hormones whose mechanisms of action are associated in part with cyclic nucleotide mediation were assessed in 37 patients with chronic azotemia. Samples were obtained prior to and immediately following conventional hemodialysis. Plasma epinephrine and norepinephrine levels in patients predialysis were increased 50% and 25% respectively compared to control subjects. Levels of insulin, prolactin, aldosterone and renin were also in creased in azotemic patients prior to dialysis. Conventional hemodialysis reduced serum levels of growth hormone, but had no effect of the elevated levels of all other hormones found in patients predialysis. In particular, plasma epinephrine and norepinephrine levels were unaffected by hemodialysis. Despite these findings, hemodialysis did reduce to normal levels the elevated plasma levels of cyclic AMP and cyclic GMP observed in uremic subjects predialysis. These data are consistent with increased adrenergic outflow in patients with chronic azotemia, and suggest a mechanism of homologous desensitization of the catecholamine receptor adenylyl cyclase unit in chronic azotemia.