Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Inhibition ofSaccharomyces cerevisiae division by 5-trifluoro-methyl-6-azauracil

Summary Cell division, as studied in asynchronous cultures of yeast cells, is sensitive to 5-trifluoromethyl-6-azauracil (F₃CAzU). Under defined conditions (10 mmoles l^–1 F₃CAzU) this compound blocks immediately and completely the process of cell division. Using synchronized cells, the time-point at which division process of yeast cell can be inhibited by F₃CAzU has been determined. The inhibitor effect of this compound is completely reversed by thymine, thymidine and uracil.     

In vitro effects of methionine-enkephalin,somatostatin and insulin on cultured gonadal cells of the snailHelix aspersa

Isolated snail gonadal cells were cultured in the presence of synthetic neuropeptides in order to determine the subsequent effect of these substances on gonadal synthetic activities. Gonadal cells were incubated for 24 h in concentrations of methionine-enkephalin, somatostatin and insulin ranging from 10^–4 M to 10^–9 M, in medium 199 supplemented with 6% Ultroser G. Synthesis of DNA and protein by the cultured cells were simultaneously estimated by measuring incorporation of³H thymidine and³⁵S methionine. The rate of labelled precursor incorporation was measured using the liquid scintillation technique. All substances tested exerted a dose-dependent effect. The synthetic activity of the cultured cells was highest when the concentration of the peptides added to the medium approximated the physiological levels. Methionine-enkephalin, somatostatin and insulin at 2×10^–8 M significantly increased³H thymidine incorporation, by 62%, 69% and 69% respectively, and protein synthesis by 42%, 57% and 57%, respectively. In the case of juvenile gonadal cultured cells, a similar increase in³H and³⁵S incorporation was registered for a 10^–7 M peptide concentration. Both lower and higher peptide concentrations inhibited³H thymidine and³⁵S methionine incorporation. Pharmacological studies suggest the existence of methionine-enkephalin and somatostatin-like receptors on snail gonadal cells. These results indicate that our gonadal cell culture model provides a useful tool for the study of the neuroendocrinological control of the activity of snail gonadal cells.     

Differential sensitivity of the two phases of ear artery contraction to intimal and adventitial norepinephrine

Summary The biphasic contraction of the rabbit ear artery to norepinephrine (NE) was investigated in the normal (adventitial stimulation) and the everted (intimal stimulation) segment of ear artery. The 2nd phase response showed an intimal ED₅₀ of 8.2×10^–8 M which was significantly (p<0.05) lower than the adventitial ED₅₀ of 42.6×10^–8 M. This difference was abolished by inhibition of neuronal and extraneuronal uptake for NE. The 1st phase response also showed an ED₅₀ for the intimal stimulation (6.9×10^–8 M) which was significantly (p<0.05) lower than adventitial (65.5×10^–8 M). This difference was reduced but not abolished by NE uptake inhibition. This suggsets that some feature of the adrenergic neuroeffector apparatus is asymmetrically arranged to favor fast responses to blood borne NE.Supported by American Heart Association-Greater Los Angeles Affiliate Grant No. 602. We wish to thank Dr John Bevan and Dr Alasdair MacLean for helpful advice.     

Growth ofNostoc muscorum mutants in the presence of diuron (DCMU) and L-methionine-DL-sulfoximine

Summary Diuron (DCMU) is inhibitory to the photoautotrophic and photoheterotrophic growth of the N₂-fixing blue-green algaNostoc muscorum at concentrations of 1.0×10^–5 M and 2.0×10^–5 M, respectively. A mutant of this organism resistant to 5.0×10^–5 M DCMU under its photoheterotrophic growth conditions, with the ability to utilize DCMU as a carbon and nitrogen source for growth, and complete inability to grow photoautotrophically has been isolated. With the apparent defect in its photosynthetic ability, it is suggested that theDCMU ^r mutant lacks the step inhibited by 1.0×10^–5 M DCMU, and metabolizes DCMU by an existing enzyme system in the absence of such inhibition. That this enzyme may be glutamine synthetase (GS) is explained with the help of a L-methionine-DL-sulfoximine (MSO)-resistant mutant ofN. muscorum which is able to grow faster with 2.0×10^–5 DCMU and is known to contain an altered GS.Thanks are due to the Council of Scientific and Industrial Research, CSIR Complex, Govt. of India, New Delhi-110012, for appointing the author to the Scientists' Pool for undertaking researches on the physiological and genetic controls of nitrogen metabolism in blue-green algae, a part of which is presented in this literature.     

Acetylcholinesterase activity in anAedes aegypti cell line

Summary Considerable acetylcholinesterase (AChE) activity was detected in anAedes aegypti established cell line. The enzyme is blocked by 10^–6 M eserine sulfate, displays excess substrate inhibition and slowly hydrolyzes butyrylthiocholine. A 2-fold stimulation of AChE activity was shown after 2 days exposure to 3×10^–7 M -ecdysone. AChE activity found in the fresh medium is the contribution of the fetal calf serum portion. A direct relationship between levels of serum and the AChE activity in the cultured cells was demonstrated.Acknowledgment. I wish to thank Dr J. Peleg of the Israel Institute for Biological Research for providing the starting culture ofAedes aegypti cells.     

Differences in luteinizing hormone release stimulated in rat anterior pituitary cells by leukotriene C4 and by gonadotropin-releasing hormone in vitro

Summary Continuous administration of leukotriene C₄ (LTC₄, 10^–10 M) to superfused rat anterior pituitary cells increased LH release for 40 min only, whereas in a parallel experiment gonadotropin-releasing hormone (GnRH, 10^–9 M) evoked a continuous increase in hormone secretion. In contrast to GnRH, LTC₄ did not desensitize rat anterior pituitary cells. The secretory action resulting from the administration of LTC₄ (10^–10 M) was abolished for 40 min after previous stimulation. The results documented the dual action of LTC₄ on LH exocytosis.     

Calcium and the contractile response to prostaglandin in the smooth muscle of guinea-pig stomach

Summary In the longitudinal smooth muscle of guinea-pig stomach, verapamil (10^–5 M) which showed marked suppression of high K-induced contractures, did not suppress the contractile response to PGE₁ (1.5×10^–9 to 10^–6 M) markedly. These results suggest that the contractile mechanism of PGE₁ in guinea-pig stomach may mainly depend on a release of bound Ca in the cell and partly depend on a Ca influx from the extracellular origin.     

Inhibition and sex specific induction of spawning by serotonergic ligands in the zebra musselDreissena polymorpha (Pallas)

Serotonin (5-hydroxytryptamine, 5-HT) stimulates spawning in the zebra mussel (Dressena polymorpha), a macrofouling European bivalve that has recently invaded North America. To develop methods of controlling zebra mussel spawning, two vertebrate serotonin antagonists, methiothepin and metergoline, known to bind with high affinity to snail 5-HT receptors, were tested for their ability to block 5-HT-induced spawning in zebra mussels. Methiothepin inhibited 5-HT-induced spawning at concentrations as low as 10^–6 M. Metergoline (10^–4 M) inhibited 5-HT-induced spawning; however, at lower concentrations (10^–8 to 10^–5 M), metergoline by itself significantly induced spawning in male, but not female zebra mussels. Metergoline (10^–5 M)-induced male spawning was inhibited by 10^–5 M methiothepin. Thus, methiothepin is the most effective inhibitor and metergoline the most powerful inducer of spawning yet tested in zebra mussels.     

Lack of effect of dihydroergotamine on endothelial and smooth muscle cell proliferation and endothelial cell prostanoid production

Summary The most important effect of dihydroergotamine is venoconstriction, but certain metabolic effects and changes in vessel prostanoid activity have also been suggested. In this study endothelial cell production of 6-keto PGF₁ and TxB₂ was quantitated in vitro. No evidence of altered prostanoid production was noted after incubation with dihydroergotamine (exposure ranging from 5×10^–3 to 5×10^–7 g/l). Similarly, no effect of dihydroergotamine on the growth rates of endothelial cells or smooth muscle cells in vitro was documented.     

Photoreactivation of UV damage in culturedDrosophila cells

Summary Cell survival and photoreactivation of 254 nm ultraviolet (UV) light damage in a wild typeDrosophila cell line was assayed by colony formation in liquid medium. F_o, F_q, and extrapolation number for the exponential portion of survival curves are 21 J/m², 3.6 J/m², and 1.5 for non-photoreactivated cells and 110 J/m², 11.2 J/m², and 1.3 for those exposed to photoreactivating light. Maximal photoreactivation occurs at the 100 J/m² region of the curve. At 10 and 50% survival, 75–80% of the UV damage was photoreactivable.     

Two phorbol ester receptor affinities in partially transformed human urothelial cells and decrease of receptor binding in desensitized cells

The presence of specific binding sites for phorbol esters was studied in a transformed but non-tumorigenic human urothelial cell line HCV-29 by assay of specific binding of³H-phorbol-12,13-dibutyrate (³H-PDBu) to intact living cells.³H-PDBu bound specifically to HCV-29 cells in a saturable and competitive manner. Scatchard plot analysis of specific binding yielded a curved plot consistent with two binding sites with K_d of 11 nM and 102 nM, respectively. At saturation the corresponding PDBu binding capacities (B_max) were 8.8 pmol/10⁶ cells (5.2×10⁶ molecules bound per cell) and 2.8 pmol/10⁶ cells (1.7×10⁶ molecules bound per cell).³H-PDBu binding was displaced by biologically active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and mezerein,but not by tumor promoters such as L-tryptophan, anthranilic acid and sodium saccharin. In cells desensitized by pretreatment with 1 g/ml (2M) TPA or PDBu for 24 h the level of binding was reduced to 28% of the level in non-exposed cells. The ability of desensitized cells to bind³H-PDBu was gradually restored within 5–6 days. At the same time the cells became sensitive to the morphological alteration induced by PDBu. This suggests that desensitization of HCV-29 cells is due to a decreased receptor-ligand binding capacity probably associated with down regulation of the phorbol ester receptors.     

Induction of the in vitro p-hydroxylation of14C-amphetamine stereoisomers in phenobarbital-treated rats

Summary The rate of p-hydroxylation of¹⁴C-(-)-amphetamine by liver microsomes was higher than that of (+)-isomer in phenobarbital-treated male rats. The apparent K_m values for (-)- and (+)-amphetamine hydroxylation were 4.54×10^–5 M and 2.27×10^–5 M respectively, in both treated and control animals.     

Evidence of a direct action of triiodothyronine (T3) on the cell membrane of GH3 cells: an electrophysiological approach

Summary Electrophysiological experiments demonstrate that triiodothyronine (T₃) exerts a direct effect on the membrane of a strain of cultured rat pituitary tumor cells, GH₃/B₆. These cells respond to pressure application of T₃ (2–5 nl, concentration 1·10^–10 M) with an increase in the membrane resistance (R_m) and a hyperpolarization. Spontaneously firing cells become silent.     

Phosphate transport inClaviceps sp. strain SD-58

Summary Phosphate uptake inClaviceps sp. strain SD-58 was found to be linear for 20 min, proportional to cell density in mg/ml, energy dependent, and taking place against a concentration gradient with a K_m value of 45.45×10^–5 M. Osmotic shock treatment to the cell caused a reduction in phosphate uptake associated with the release of binding protein. Partial restoration of uptake was observed on incubation of osmotically shocked cells with shock fluid. The results are discussed with reference to the effect of phosphate on alkaloid synthesis inClaviceps sp. strain SD-58.     

Modification of theophylline-induced lipolysis in human fat cells after trypsination

Summary Trypsin-treatment of human fat cells results in the potentiation of the lipolytic response and the cAMP accumulation induced by theophylline (5·10^–4 M) but not of those induced by theophylline (5·10^–3 M). The amount of cAMP formed after exposure to theophylline (5·10^–3 M) plus norepinephrine (5·10^–6 M) remains, however, 2.6fold higher in trypsin-treated human fat cells than in the control ones.Acknowledgments. The authors gratefully acknowledge the help of the surgical staff of the C. H. I. of Poissy. This work was supported by grants from the C. H. I. of Poissy and from the Université René Descartes.     

Retinoic acid enhances the proliferation of smooth muscle cells

Summary Retinoic acid (RA, 10^–5–10^–7 M) is shown to enhance the proliferation of cultured rat aortic smooth muscle cells (SMC). This effect is not connected with a synergistic action of RA together with serum mitogens. Moreover, the expression of L1, a surface antigen specific for modulated SMC entering the cell cycle, is amplified by RA treatment.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Phosphoramidon enhances allatostatin-mediated inhibition of juvenile hormone biosynthesis in the corpora allta of the cockroach,Diploptera punctata

Use of the enkephalinase inhibitor phosphoramidon in the in vitro radiochemical assay for juvenile hormone biosynthesis enhanced allatostatin-mediated inhibition of hormone production by corpora allata of the cockroach,Diploptera punctata. Significant increases in inhibition in day 2 virgin female CA by AST 1 (at 10^–7 M) and AST 4 (10^–8–10^–7 M) were observed in the presence of phosphoramidon (10^–5M or greater). No significant increases in inhibition were seen in CA from day 6 mated females with AST 4 (10^–9–10^–7M) and phosphoramidon combined. Phosphoramidon alone had no effect on JH biosynthesis. Analysis of allatostatin content of the CA, as determined by ELISA, revealed that addition of phosphoramidon to the medium increased the endogenous allatostatin conten in CA of virgin and mated females. The similarity in primary structure between allatostatins and enkephalin-like peptides and their similar distribution makes it probable that phosphoramidon acts by preventing breakdown of allatostatins within the CA.     

The effect of perchlorate ion and some pharmacological agents on the sensitivity of the frog (Rana pipiens) rectus abdominis to acetylcholine

Zusammenfassung Die Reaktion des mit Eserin vorbehandelten musculus rectus abdominis des Frosches auf Acetylcholin liess sich durch Oxotremorin (2.0·10^–5 M), Arecolin (2.0·10^–5M), Aceclidin (1.0·10^–4 M) und Carbachol (2.0·10^–7 M) verstärken, während Atropin (1.0·10^–5 M) hemmend wirkte. Perchlorationen in Konzentrationen über 2 · 10^–4 M sensibilisierten den Froschmuskel erheblich und reduzierten die Grenzdosis, bei der eine Potenzierung durch Substanzen mit muskarinähnlicher Wirkung beobachtet wird. Alle beschriebenen Reaktionen liessen sich durch Gehirnextrakte verstärken, in denen zuvor Acetylcholin durch Kochen in alkalischer Lösung zerstört worden war.

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This work was supported by NIH Grants No. NB 03007 and No. 5-F1-GM-22,905.