Isoproterenol-induced selective phosphorylation invivo of the 214,000 dalton subunit of rat C6 glioma cell RNA polymerase II

The phosphorylation $\text{in}\text{vivo}$ of RNA polymerase II after isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Glioma cells were incubated in the presence of Na₂H³²PO₄ and stimulated for 1 hour with the β-adrenergic agonist isoproterenol. The phosphorylation pattern was analyzed after purification of RNA polymerase II by immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. Isoproterenol markedly increased [³²P]phosphate incorporation into the 214,000 dalton RNA polymerase subunit. Analysis of the phosphate acceptor amino acid revealed the presence of only [³²P]phosphoserine. The data demonstrates an isoproterenol-induced structural modification of RNA polymerase II.     

Phosphorylation of chromosomal proteins as a function of age and its modulation by calcium

$\text{In vitro}$ phosphorylation of histones and non histone chromosomal proteins (NHCP) and its modulation by calcium have been studied using slices of cerebral hemisphere of rats of various ages. Phosphorylation of histones decreases, and that of NHCP increases with increasing age. Calcium inhibits phosphorylation of histones of young and adult rats, but stimulates phosphorylation of NHCP. Phosphorylation of H1 and H4 histones is greater than that of other histones, and calcium inhibits their phosphorylation more markedly than of other histones. The significance of such modifications of chromosomal proteins in the aging process is discussed.     

Extracellular magnesium is not required for beta adrenergic drug-receptor interactions in intact human lymphocytes

We have investigated the effect of extracellular magnesium ions on the function of beta adrenergic receptors in $\text{intact}$ human lymphocytes. We examined adenylate cyclase stimulation by isoproterenol displacement of (-) (³H) dihydroalprenolol from beta receptor sites, and down regulation (desensitization) of beta receptors by prolonged exposure of the cells to isoproterenol. Contrary to results obtained using broken cell preparation, in none of these situations did the presence or absence of extracellular magnesium ions make any difference. The importance of selecting the most nearly physiological preparations for conducting $\text{in vitro}$ studies that may be extrapolated to the whole organism is stressed.     

Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).     

Butyric acid: A small fatty acid with diverse biological functions

Butyric acid, a 4-carbon fatty acid, affects morphology, growth rate and gene expression in mammalian cells in culture. Sodium butyrate (0.5 to 3 mM) produces reversible growth inhibition in several mammalian tumor cells in culture, but it causes cell death only in human neuroblastomas and human glioma cells in culture. Sodium butyrate in combination with currently used tumor therapeutic agents produced a synergistic, an additive or no effect on growth of mouse neuroblastoma cells and rat glioma cells in culture. At least in NB cells, the cell death and growth inhibition may be related to the reduction in anaerobic glycolysis. Sodium butyrate increases the expression of one or more differentiated functions in mouse NB cells, mouse erythroleukemic cells, human epidermoid carcinoma, human colon carcinoma cells and Chinese hamster ovary cells. The induction of differentiation by butyrate may in part be related to an increase in the cellular cyclic AMP level. Sodium butyrate increases the activities of several enzymes, whereas, it decreases the activities of some. The increase of some enzymes appears to be correlated to hyperacetylation of histones. $\text{In}$$\text{vitro}$ studies suggest that sodium butyrate may be useful in the management of neoplasms by causing selective cell death, and/or cell differentiation and by increasing the cell killing effect in conjuction with currently used tumor therapeutic agents. Sodium butyrate can also be used as a tool to study the regulation of gene expression in mammalian cells.     

Effects of polyamines on in vitro phosphorylation and acetylation of histones of the cerebral cortex of rats of various ages

$\text{In}\text{vitro}$ phosphorylation and acetylation of histones and their modulation by spermine and spermidine were studied using slices of cerebral cortex of female rats of various ages. Phosphorylation and acetylation of individual histones decrease with increasing age. Spermine and spermidine have stimulatory effects on both the modifications of specific histones in immature rats. These effects decrease with increasing age. Such changes in covalent modifications of histones may alter gene expression and contribute to the aging process.     

Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular mucin of the mouse

The influence of isoproterenol and pilocarpine on the in vitro incorporation of [³H]leucine and $\text{N-}\text{acetyl}\text{[}{}^{14}\text{C}\text{]}\text{mannosamine}$ into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [³H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [¹⁴C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [¹⁴C]ManNAc relative to [³H]leucine into submandibular mucin of the mouse. During a period of 10 h the [¹⁴C]ManNAc incorporation into the mucin was enhanced 2–3-fold by isoproterenol and 3–4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.     

Chemical modification of mitochondria. II. Effect of labeling on oxidative phosphorylation

The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10^?5m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10^?4m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the $\text{P}\text{O}$ ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The $\text{P}\text{O}$ ratio increases and tends to level off as the incubation time increases. No increase in $\text{P}\text{O}$ ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process.     

Histone phosphorylation in phorbol ester stimulated and β-adrenergically stimulated mouse epidermis in vivo and characterization of an epidermal protein phosphorylation system

Under certain physiological conditions a change i n the phosphorylation of histones in mouse epidermis in vivo was observed. Thus a single local application of the tumor-promoting mitogen 12-O-tetradecanoylphorbol-13-acetate caused a long-lasting increase of histone H1 phosphorylation which paralleled stimulated cell proliferation. Injection of the antimitotic β-adrenergic agonist isoproterenol led to a temporatory decrease in the rate of phosphorylation of H1, H2A and H2b immediately after cyclic AMP accumulation. A complete protein phosphorylation system could be demonstrated in mouse epidermis homogenates. The following enzyme activities were partially purified and characterized: a cyclic AMP-dependnet histone kinase; a ‘casein kinase’ and an ‘unsopecific’ protein kinase; a histone-specific protein phosphatase; and two ‘unspecific’ phosphoprotein phosphatases. In addition, a stimulatory effect of cyclic GPM on histone phosphorylation was observed. The enzymes were found to be predominantly localized in the 105 000 × g supernatant, but a small proportion of protein kinase and phosphatase activity could be regularly demonstrated in cell nuclei.     

Opposite effects of acute and repeated administration of desmethylimipramine on adrenergic responsiveness in rat pineal gland.

The effect of acute and repeated desmethylimipramine (DMI) treatment on catecholamine-stimulated production of adenosine 3', 5'-monophosphate (cyclic AMP) in rat pineal gland was studied $\text{in}$$\text{vivo}$. In rats exposed to continuous illumination, the administration of isoproterenol (2μmol/kg) to control animals produced a marked increase in the concentration of cyclic AMP in pineal gland. In contrast, norepinephrine (2μmol/kg) failed to increase the levels of cyclic AMP. After acute treatment with DMI (single injection, 38μmol/kg, i. p.), the isoproterenol-induced rise in cyclic AMP was not significantly different from that measured in control animals. However, acute DMI treatment did allow a significant elevation in the concentration of cyclic AMP in pineal gland in response to norepinephrine. In rats given nine injections of DMI (38μmol/kg, i.p., twice daily) neither isoproterenol nor norepinephrine caused a significant increase in the concentration of cyclic AMP in pineal glands. Although acute treatment with DMI had no significant effect on [³H] dihydroalprenolol binding, chronic treatment with DMI significantly reduced [³H] dihydroalprenolol binding in the pineal gland. The results of this study suggest that while a single administration of DMI can enhance adrenergic responses elicited by norepinephrine, chronic administration of DMI leads to compensatory decreases in receptor density and adrenergic responsiveness.     

Effect of ethanol upon isoproterentol stimulation of growth and secretion in the mouse parotid gland

Isoproterenol (0.3 mmole/kg body wt.), when injected into the mouse intraperitoneally, increases the weight by 35% and stimulates DNA synthesis 30-fold in the parotid gland. The induction of both hypertrophy and hyperplasia is completely inhibited by ethanol at a dose of 200 mmole/kg body wt. but is almost unaffected by 60 mmole/kg. The full inhibiton of both growth parameters is observed when ethanol is administered up to 5 hr after isoproterenol. Partial inhibition is observed when ethanol is given as long as 15 hr after isoproterenol. It contrast ethanol did not alter the secretion of α-amylase in response to isoproterenol. Ethanol had no effect upon the rise in cyclic GMP level caused by isoproterenol but augmented the rise in cyclic GMP In agreement with these $\text{in}$$\text{vivo}$ observations, low concentrations of ethanol activated adenylate cyclase $\text{in}$$\text{vitro}$, however guanylate cyclase activity was quite strongly inhibited. Although high levels of ethanol (300 mmole/kg) inhibited the induction of both ornithine decarboxylase and S-adenosylmethionine decarboxylase little inhibition was seen at 200 mmole/kg suggesting that the interference with polyamine metabolism is not the mechanism of the ethanol effect upon isoproterenol-induced parotid growth.     

Polyamine induced changes in the ADP-ribosylation of nuclear proteins from rat liver

Purified rat liver nuclei were incubated $\text{in vitro}$ with [³H]NAD. Altered patterns of ADP-ribosylation of nuclear proteins occurred with 1 mM spermidine or spermine with the latter polyamine causing the greater change. Spermine treated nuclei showed a two-fold increase in ADP-ribose incorporation into H1 histones and a decrease in the other histones. Likewise, the incorporation into the more acidic non-histone nuclear proteins was greater with spermine than spermidine. These results suggest that polyamines may exert a regulatory function by altering the pattern of ADP-ribosylation of both histone and non-histone nuclear proteins.     

Modulation by the ratio S-adenosylmethionineS-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. $\text{S-}\text{Adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio $\text{S-}\text{adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ can modulate phosphorylation of a specific protein.     

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

Rapid modulation by progesterone and tamoxifen of estradiol effects on nuclear histone acetylation in the uterus of the fetal guinea pig

The effect of estradiol, progesterone, tamoxifen, estradiol + progesterone or estradiol + tamoxifen on the [³H]acetylation of histones in the fetal uterus of guinea pig was studied. The fetuses were injected subcutaneously ‘in situ’ with the hormones or $\text{tamoxifen}\text{+ [}{}^3\text{H}\text{]}\text{acetate}$ alone. In 10 min, estradiol stimulated the acetylation of histone 10–12-times with respect to the control animals. Progesterone and tamoxifen blocked this effect. It is suggested that histone acetylation is an early step induced by estrogen action during intrauterine life and that progesterone and tamoxifen suppress this mechanism very effectively.     

Topoisomerase inhibitor induced dephosphorylation of H1 and H3 histones as a consequence of cell cycle arrest

Posttranslational modifications of histones have an integral function in the structural and functional organization of chromatin. Several changes in the modification state of histones could be observed after induction of apoptosis with topoisomerase inhibitors and other inducers. Most of these studies include the analysis of the state of phosphorylation of histones, and the results are to some extent controversial, depending on cell lines and agents used. In the present study we compared the kinetics of the dephosphorylation of H1 and H3 histones with apoptosis markers after treatment of leukemic cell lines with topoisomerase inhibitors. In parallel, we determined cell cycle parameters in detail. Dephosphorylation of both histone classes started within 1 h of induction, and no direct correlation with timing and intensity of the investigated apoptotic features could be observed. In contrast, we show that the effect of topoisomerase inhibitors on the state of H1 and H3 phosphorylation is not directly related to apoptosis, but reflects the changes in the cell cycle distribution of cells treated with these inducers.     

Influence of isoproterenol on net potassium uptake in whole pigeon erythrocytes in vitro

Isoproterenol increases net uptake of potassium in whole pigeon erythrocytes $\text{in vitro}$; effect of 10^?5 M isoproterenol is blocked by 10^?4 M propranolol. Pentifylline, a potent inhibitor of cAMP-phosphodiesterase, significantly amplifies effect of isoproterenol, indicating that isoproterenol-effect is mediated by cAMP. cAMP alone has no direct influence on net potassium uptake, while dibuturyl-cAMP has a very weak effect. Isoproterenol-effects are also mediated by the cell membrane protein-phosphorylation.     

Inhibition by dexamethasone of the in vitro transport of 3-O-methylglucose into rat thymocytes

Reduced glucose transport across the plasma membrane and reduced phosphorylation may both be responsible for the early inhibitory effect of physiological concentrations of glucocorticoids on glucose uptake by rat thymocytes.The early inhibitory effects of glucocorticoids (5 · 10^?7 M dexamethasone) on glucose consumption and ¹⁴CO₂ formation from d-[U-¹⁴C]glucose were reproduced.The total uptake curve of 4.8 μM $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ was biexponential with $\text{t}\text{1}\text{2}$ of 1.1 min and 36 min, respectively, the rapid part comprising about 50% of the equilibrated intracellular water space. The latency of the effect of 5 · 10^?7 M dexamethasone on $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ uptake ranged from 15 to 100 min and the inhibition varied from 15 to 55% independently of the lag period. The effect of 3-O-methylglucose concentration on the initial uptake by steroid-responsive cell preparations was tested after 45 min of preincubation with or without 5 · 10^?7 M dexamethasone. In 12 experiments dexamethasone reduced V from 1.36 ± 0.16 mmol · min^?1 · l^?1 cell water to 0.81 ± 0.10 mmol · min^?1 · l^?1 cell water with insignificant change of K_m (6.0 mM versus 5.9 mM). Dexamethasone had similar effect after 90 or 120 min.The variabilities of control cell transport capacity, the lag period and the magnitude of the dexamethasone effect could not be accounted for by changes in pH, effects of cell density, concentrations of albumin, ethanol, nucleosides, pyruvate or correlated to age and sex of the rats. In conclusion the inhibition of glucocorticoids on glucose consumption by thymocytes appears to be an inhibited plasma membrane transport capacity.     

Carnitine-induced uptake of l-carnitine into cells from an established cell line from human heart (CCL 27)

l-Carnitine is actively transported into Girardi human heart cells, an established cell line from human heart. The present study was undertaken to investigate the effect of different concentrations of l-carnitine in the growth medium on the rate of uptake of l-[³H]carnitine.Increasing the concentration of l-carnitine from 2 to 100 μmol/1 in the growth medium of the cells, increased the rate of uptake of l-[³H]carnitine by about 50%. The maximal effect was reached after approx. 72 h incubation. The increase in rate seemed to be caused by synthesis of increased number of carriers, as judged by the increase in $\text{V}$ with unchanged apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for the transport process. This effect of l-carnitine could be inhibited by cycloheximide, indicating the dependence on intact protein synthesis. The morphology of the cells was studied by electron microscopy. No myofilaments were found, thus the cells are dedifferentiated and no longer typical muscular cells.