E. Keck

β-Adrenergic agonist- and prostaglandin-mediated regulation of cAMP levels in Ewing's sarcoma cells in culture

This is the first report demonstrating hormonal control of cAMP levels in Ewings sarcoma cells. Two classes of hormones, beta-adrenergic agonists and prostaglandins stimulate cAMP production in cultured Ewings sarcoma cells. The efficacy order for beta-adrenergic agonists is (-)-isoprenaline greater than or equal to (-)-noradrenaline greater than or equal to (-)-adrenaline much greater than (-)-phenylephrine. The stimulatory effect of (-)-noradrenaline is antagonized by beta 1-selective metoprolol and also by beta 2-selective ICI 118,551. The efficacy order for prostaglandins (PG) is PGE1 greater than PGI2 greater than PGE2 much greater than PGF2 alpha; 6-keto PGF1 alpha and PGD2 do not influence cAMP levels in Ewings sarcoma cells. Cultures pretreated with PGE2 are less responsive to a second challenge with PGE2 but their response to (-)-isoprenaline is unimpaired. Similarly, pretreatment with (-)-isoprenaline induces homologous desensitization to (-)-isoprenaline and has no effect on the PGE2-stimulated increase in cAMP. We conclude that these cells provide an ideal model for the study of the initial steps of beta-adrenergic and prostaglandin action in Ewings sarcoma.

Forskolin inhibition of glucose transport in bone cell cultures through a cAMP-independent mechanism

Studies are presented demonstrating inhibition of glucose transport by forskolin in human MG-63 osteosarcoma cells as well as osteoblast-like cells derived from normal human trabecular bone and chick calvaria. The cAMP-stimulators parathyroid hormone, prostaglandin E2, and isoproterenol did not influence glucose transport. Benzyl alcohol, a membrane lipid fluidity modulator, also provoked inhibition of the glucose uptake rate. Effects of forskolin and benzyl alcohol were not additive. It is suggested that cAMP is not a mediator of glucose transport in bone cells, and that forskolin inhibits glucose transport via a cAMP-independent mechanism.

Neuropeptide Y inhibits vasoactive intestinal peptide‐ and dopamine‐induced cyclic AMP formation in human Ewing's sarcoma WE‐68 cells

Neuropeptide Y (NPY) regulation of intracellular cyclic AMP accumulation was studied in human Ewings sarcoma cell line, WE‐68. NPY inhibited vasoactive intestinal peptide (VIP)‐ and dopamine‐stimulated but not basal cyclic AMP formation. The peptide effect was rapid (<2 min), concentration‐dependent with a half‐maximal effective concentration (EC50) of 8 nM NPY, and maximal inhibition reaching 60–70% with 100 nM NPY. Prior exposure of WE‐68 cells to pertussis toxin completely abolished the inhibitory action of NPY. It is concluded that NPY attenuates agonist‐stimulated cyclic AMP formation in Ewings sarcoma WE‐68 cells, and may do so via the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.

Induction of glycogenolysis in cultured Ewing's sarcoma cells by dopamine and β-adrenergic agonists

SummaryThis study describes hormonal regulation of glycogen metabolism in Ewings sarcoma cells. 3H-Glycogen synthesized in cultured Ewings sarcoma WE-68 cells from 3H-glucose was hydrolyzed in a concentration-dependent manner by various catecholamines. The order of potency for the glycogenolytic effects of catecholamines was isoproterenol ≧ dopamine > norepinephrine > epinephrine. The concentrations giving half-maximal effectiveness (EC50) were about 2×10-8M, 3×10-8M, 8×10-8M, and 5×10-7M for isoproterenol, dopamine, norepinephrine, and epinephrine, respectively. Higher concentrations of each of the catecholamines were necessary to elicit EC50 stimulation of cyclic AMP production in Ewings sarcoma cells. Glycogenolysis induced by dopamine was blocked by chlorpromazine, a dopamine D1-receptor antagonist, but not by haloperidol, a dopamine D2-receptor antagonist. The glycogenolytic action of norepinephrine was markedly reduced by propranolol, a β-adrenoreceptor antagonist, and was not affected by yohimbine, an α-adrenoreceptor antagonist. In addition, chlorpromazine also antagonized the glycogenolytic response to norepinephrine. Dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, and the diterpene forskolin were also found to induce 3H-glycogen hydrolysis. Our data indicate that cate-cholamines exert their glycogenolytic effects in Ewings sarcoma cells by stimulation of cyclic AMP formation via β-adrenergic recepetors and dopamine D1-receptors.

Vasoactive intestinal peptide receptor regulation of cAMP accumulation and glycogen hydrolysis in the human Ewing's sarcoma cell line WE-68

This study describes functional characteristics of receptors for vasoactive intestinal peptide (VIP) on human Ewings sarcoma WE-68 cells. These characteristics include 125I-VIP binding capacity, cellular cAMP generation, glycogen hydrolysis, and pharmacological specificity. Binding studies with 125I-VIP showed specific, saturable, binding sites for VIP in WE-68 cells. Scatchard analysis revealed the presence of a single class of high-affinity binding sites that exhibited a dissociation constant (Kd) of 90 pM and a maximal binding capacity (Bmax) of 24 fmol/mg of protein. VIP and VIP-related peptides competed for 125I-VIP binding in the following order of potency: human (h) VIP greater than human peptide with N-terminal histidine and C-terminal methionine (PHM) greater than chicken secretin much greater than porcine secretin. Glucagon and the C-terminal fragments VIP[10-28] and VIP[16-28] and the VIP analogue (D-Phe2)VIP did not inhibit 125I-VIP binding. Addition of hVIP to WE-68 cells provoked marked stimulation of cAMP accumulation, hVIP stimulated increases in cAMP content were rapid, concentration-dependent, and potentiated by 3-isobutyl-l-methylxanthine (IBMX). Half-maximal stimulation (EC50) occurred at 150 nM hVIP. The ability of hVIP and analogues to stimulate cAMP generation paralleled their potencies in displacing 125I-VIP binding. (D-Phe2)VIP, VIP[10-28], VIP[16-28], and (p-Cl-D-Phe6, Leu17)VIP, a putative VIP receptor antagonist, affected neither basal cAMP levels nor hVIP-induced cAMP accumulation. WE-68 cell responses to hVIP were desensitized by prior exposure to hVIP. Desensitization to hVIP did not modify the cAMP response to beta-adrenergic stimulation, and beta-adrenergic agonist desensitization did not modify responses to hVIP. hVIP also induced a time- and concentration-dependent hydrolysis of 3H-glycogen newly formed from 3H-glucose in WE-68 cultures. hVIP maximally decreased 3H-glycogen content by 36% with an EC50 value of about 8 nM. The order of potency of structurally related peptides of hVIP for stimulation of glycogenolysis correlated with their order of potency for inhibition of 125I-VIP binding. IBMX potentiated the glycogenolytic action of hVIP and PHM. The simultaneous presence of the calcium channel antagonist verapamil or the calcium ionophore A 23187 did not influence the glycogenolytic and cAMP stimulatory effects of hVIP. Collectively, these data indicate that Ewings sarcoma (WE-68) cells are endowed with genuine VIP receptors which are coupled to the formation of cAMP that probably serves a second messenger role in stimulating glycogen hydrolysis in these cells in response to VIP.(ABSTRACT TRUNCATED AT 400 WORDS)

An Immunocytochemical and Biochemical Study of Ewing’s Sarcoma Cells in Culture: Evidence for Neural Differentiation in Vitro

Ewing’s sarcoma, a primary bone tumor of childhood and adolescence, was first described more than 65 years ago yet the biological characteristics of this malignant tumor have remained undetermined. Morphologically Ewing’s sarcoma is often indiscernible from other small round-cell tumors such as neuroblastoma, rhabdomyosarcoma and lymphoma-leukemia. In conseguence, the cellular origin of Ewing’s sarcoma is still subject to debate. Whereas Ewing himself postulated an endothelial origin for this tumor (Ewing 1921), most recent authors suggest that Ewing’s tumor is of mesenchymal derivation (Dickman et al. 1982, Miettinen et al. 1982, Navas-Palacios et al. 1984, Scarpa et al. 1987). Recently, van Valen et al. (1987) demonstrated β-adrenergic agonist- and prostaglandin-mediated stimulation of intracellular cyclic AMP production in a Ewing’s sarcoma cell line, WE-68, and homologous desensitization of the cyclic AMP response of these cells to isoproterenol and PGE2. In the present study we show that dopamine also stimulates cyclic AMP levels and promotes hydrolysis of newly synthesized 3H-glycogen in Ewing’s sarcoma WE-68 cultures. In addition, Ewing’s sarcoma cells were found to immunostain positively to antibodies detecting antigens that are specific markers for nervous system cell elements or are coexpressed in neural cell types.

Effects of Pertussis Toxin on the Regulation of Human Osteosarcoma Adenylate Cyclase by Parathyroid Hormone, Isoproterenol, Prostaglandin E2, andForskolin

Regulation of adenylate cyclase is accomplished by transmission of signals from the agonist-occupied receptor to the catalytic moiety of adenylate cyclase via guanine nucleotide-binding regulatory components (1). Hormone-induced stimulation is mediated by a stimulatory guanine nucleotide-binding regulatory protein (Ns). Ns is a heterotrimer with 45 kDa (α), 35 kDa (β), and 10 kDa (γ) subunits (2). The α-subunit of Ns possesses a site for guanine nucleotide binding and has GTPase activity that can be inhibited by ADP-ribosylation catalyzed by cholera toxin (3). Attenuation of adenylate cyclase activity by hormones, GTP, and stable GTP-analogs appears to be mediated by an inhibitory guanine nucleotide-binding regularory protein (Ni); this protein also has a heterotrimeric structure with 41 kDa (α)t 35 kDa (β), and 10 kDa (γ) subunits (4, 5). The α-subunit of Ni also binds guanine nucleotide and is likewise a GTPase (6). The α and β subunits of Ns and Ni are physically and probably functionally identical (7). Pertussis toxin, also called islet-activating protein, has been shown to modulate the adenylate cyclase system in several intact cell and membrane preparations. Following exposure of cells to pertussis toxin, agonist-mediated inhibition of adenylate cyclase was attenuated (8) while the responsiveness of adenylate cyclase to stimulatory hormones was enhanced (9). The effects of pertussis toxin appeared to coincide with an ADP-ribosylation of the a-subunit of Ni, inactivating Ni (10).

Regulation of Amino Acid Transport and Protein Synthesis by 1,25-Dihydroxyvitamin D3 and Dexamethasone in Chick Osteoblasts

As both 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (Dexa) are known to influence osteoblastic activity (stimulation and inhibition of alkaline phosphatase and growth), we examined the effect of the two steroids on the amino acid transport across the membrane of chick osteoblasts on the 4th and 6th day in growing cultures.

Inhibition of Protein Synthesis and Amino Acid Transport by Dihydrotestosterone and 17ß-Estradiol in Chick Osteoblasts

Although androgens and estrogens appear to be important regulators of skeletal growth, little is known about their direct effects on bone cells i.e. osteoblasts. Burnett et al. (1983) report about a significant increase in 3H-thymidine incorporation per µg DNA in bone. Canalis et al. (1978) showed that 17β-estradiol (E2) had a small and inconsistent stimulatory effect on the incorporation of 3H-proline into collagenase-digestible protein in rat calvariae and testosterone had no effect. In order to evaluate the potency of E2 and dihydrotestosterone (DHT) on protein synthesis and amino acid (AA) transport in osteoblast, we examined their effects in net AA uptake in chick osteoblasts (OB) on the 4th and 6th day in growing cultures.