Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts.

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilatation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts

Summary The presence of specific binding sites for [³H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (K_D=31.0 nM) and low (K_D=6.05 M) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in boththe cytoplasm and the nuclei.     

Thiamine-binding activity ofSaccharomyces cerevisiae plasma membrane

Summary The specific binding activity to [¹⁴C]thiamine was found to be located in the plasma membrane ofSaccharomyces cerevisiae. The activity was inhibited by several thiamine analogs and it was hardly detectable in the plasma membrane from a thiamine transport mutant ofSaccharomyces cerevisiae. Some properties of the thiamine-binding activity of yeast plasma membrane are discussed in connection with those of the thiamine transport system.     

On the binding of steroid sulfates to albumin

Summary ³H-Labeled steroid sulfates, sulfate of estrone (E₁S) or dehydroepiandrosterone (DHAS), were dialyzed against delipidated human serum albumin or human plasma in the presence of increasing amounts of competing non-labeled sulfates (DHAS or E₁S). The apparent equilibrium constants (K) of the tracers did not measuraby change at concentrations of the non-radioactive sulfates below 10^–5 mol/l. At higher concentrations, K decreased gradually. The apparent equilibrium constant of³H-E₁S was diminished by plasma in a similar fashion. It may be concluded that albumin possesses one strong, non-specific binding site. This site, however, does not seem to be utilized for the binding of E₁S in vivo, because of its preferetial occupation by other ligands. This may be true for other steroid sulfates as well, depending on their relative abundance in plasma.Acknowledgment. This investigation received financial support from the Special Programme of Research, Development and Research Training in Human Reproduction, World Health Organisation, and from the Ford Foundation.     

Decrease in [3H]ouabain binding sites in heart and brain from spontaneously hypertensive rats

Summary A decrease in the number of binding sites (B_max) for [³H]ouabain was observed in the heart (37%) and the brain (22%) of spontaneously hypertensive rats (SHR) when compared with age-matched control Wistar Kyoto rats. No variation was detected in the affinity constant (K_D).     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group

Summary (1 R) [1-³H,²H₁] 3-Phenylpropanol, the key intermediate in the synthesis of (4 R) [4-³H,²H₁] D, L-homoserine and of the (4 S)-isomer, is obtained from (1 S) [1-²H₁] 3-phenylpropanol and (1 RS) [1-³H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD⁺; under similar conditions 2-phenylethanol undergoes very small exchange with [1-²H₂] ethanol.     

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

Summary The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [³H]inositol monophosphate, [³H]inositol bisphosphate and [³H]inositol trisphosphate. This effect was prevented by 5-HT₂ specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT₂ receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.Part of the data contained in this paper was presented at the 74th local meeting of the Japanese Society of Pharmacology at Kanagawa.     

Binding of matrilysin-1 to human epithelial cells promotes its activity

Matrix metalloproteinase-7 (MMP-7, matrilysin- 1) modulates crucial biological events by processing many epithelial cell surface-associated effectors. We addressed MMP-7 interaction with human epithelial cells and its resulting activity. In human endometrium, a model of controlled tissue remodeling, proMMP-7 was diffusely immunolocalized inside epithelial cells, whereas MMP-7 delineated their entire plasma membrane. Endometrial explants preferentially retained active MMP-7, but not proMMP-7. Endometrial epithelial cells and carcinoma cells from various tissues bound active MMP-7. Endometrial carcinoma-derived Ishikawa cells showed high affinity (K_D of ~2.5 nM) and capacity (~260 000 sites per cell) for MMP-7. MMP-7 binding decreased by extracting membrane sterols or interfering with heparan sulfate proteoglycans, and was abrogated by tissue inhibitors of metalloproteinase-2 (TIMP-2) or synthetic MMP inhibitors. Bound MMP-7 not only remained fully active towards a macromolecular substrate but also became resistant to TIMP-2. We conclude that MMP-7-selective targeting to the plasma membrane of epithelial cells promotes its activity by conferring resistance to TIMP-2. A. Berton, C. Selvais: These authors contributed equally to this work. P. J. Courtoy, E. Marbaix, H. Emonard: These authors contributed equally to the supervision of this work. Received 20 September 2006; received after revision 30 November 2006; accepted 18 January 2007     

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts

The presence of specific binding sites for [3H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (KD = 31.0 nM) and low (KD = 6.05 microM) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in both the cytoplasm and the nuclei.     

Effect of various solubilizers on angiotensinII receptors in bovine adrenocortical plasma membranes

Summary 3 monionic detergents, Triton X-100, Lubrol WX and NP-40, inhibited binding of [³H]-AT_II to bovine adrenocortical plasma membranes. This effect appeared to be direct and not due to solubilization of the AT_II receptor by these agents. Sodium deoxycholate and the chaotropic ions, ClO ₄ ^– and Br^–, produced effects similar to the nonionic detergents.Supported by grants from the Quebec Heart Foundation and the Medical Research Council of Canada. The authors acknowledge the technical assistance of Mrs D. Michaud and the secretarial aide of Mrs D. Huot-Blais, and Miss L. Leblanc.     

Histamine binding to H2 receptors stimulates phospholipid methylation in mast cells

Summary Rat peritoneal mast cells incubated in vitro in the presence of L-[methyl-³H] methionine and exposed to histamine undergo a rapid but transient increase in phospholipid methylation. By using specific H₁- and H₂-receptor antagonists, and histamine analogues differing in their H₂-receptor agonist potency, it has been demonstrated that this metabolic event is dependent on histamine binding to H₂-receptors.We are grateful to Smith Kline and French Laboratories for the generous gift of histamine analogues.     

The role of calcium in aggregation and development ofDictyostelium

Changes in cytosolic Ca²⁺ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca²⁺]_i) at a low level in the face of steep gradients of extracellular and sequestered Ca²⁺. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca²⁺ into the cytosol and elevation of [Ca²⁺]_i. After such stimulation Ca²⁺ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca²⁺]_i to its basal level. Such responses to elevation of [Ca²⁺]_i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.     

Drosophila melanogaster does not dealkylate [14C]sitosterol

Summary Drosophila melanogaster was unable to dealkylate and convert [¹⁴C]sitosterol to cholesterol and no evidence was found for conversion of [¹⁴C]desmosterol to cholesterol. Therefore,D. melanogaster is incapable of dealkylating and converting C₂₈ and C₂₉ phytosterols to cholesterol.The authors thank O. J. Duncan III, and H. S. Symonds for technical assistance. Mention of a company name or proprietary product does not imply endorsement by the U.S. Department of Agriculture.     

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.     

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N₃-[-³²P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C₄ (LTC₄) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N₃-[-³²P]ADP trapping, weakened inhibition of LTC₄ transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.Received 1 May 2003; received after revision 18 June 2003; accepted 24 June 2003     

Variations au cours de la journée de l'incorporation in vivo de la leucine tritiée dans les protéines du cervelet et du cerveau du jeune rat normal et hypothyroïdien

Summary In the normal and hypothyroid 6-day-old rat, the specific radioactivity (RSA) and the relative RSA (ratio of the RSA to the [³H] leucine concentration of the acido soluble phase) of the cerebral and cerebellar proteins, change during the day synchronally. They show a maximum at 15.00 h and a minimum at 03.00 h. At all stages studied, these values are significantly lower in the hypothyroid animals than in normal ones.     

3[H]-Sulpiride labels mesolimbic non-dopaminergic sites that bind antidepressant drugs

3[H]-(-)-Sulpiride and 3[H]-spiperone binding was compared in rat amygdala, nucleus accumbens and striatum, using (+/-)-sulpiride to define specific binding. 3[H]-(-)-Sulpiride bound to twice as many sites in amygdala and nucleus accumbens as 3[H]-spiperone. 3[H]-(-)-Sulpiride binding was directed to these additional sites by using 1 microM spiperone to mask dopaminergic binding. The binding of 3[H]-(-)-sulpiride to these sites was high affinity, reversible, Na+-dependent, but not stereospecific. Metoclopramide, tiapride and antidepressant medications, but not other neuroleptics, ADTN, or serotonin displaced 3[H]-(-)-sulpiride binding to these sites. These data suggest that 3[H]-(-)-sulpiride labels mesolimbic sites other than dopamine receptors which may mediate antidepressant effects.     

High affinity recognition of a <Emphasis Type="Italic">Phytophthora</Emphasis> protein by <Emphasis Type="Italic">Arabidopsis</Emphasis> via an RGD motif

The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [¹²⁵I]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53–56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp⁵⁶ into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.Received 23 October 2003; received after revision 5 December 2003; accepted 12 December 2003     

A2 adenosine receptors in human glomerular mesangial cells

A₂ adenosine receptors were characterized in human glomerular mesangial cells using [³H] 5-N-ethylcarboxamidoadenosine (NECA) as a tracer. There was a single group of receptor sites with a K_D of 184 nM, and a number of sites of 317 fmol/mg of cell protein. Adenosine agonists increased 5-nucleotidase activity via A₂ receptor stimulation. The specific A₂ agonist-NECA, at 0.1 and 1 m, was a potent inhibitor of DNA synthesis.     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998