Odile Durieu-Trautmann

Are Several G Proteins Involved in the Different Effects of Endothelin-1 in Mouse Striatal Astrocytes?

Abstract: High‐affinity specific receptors of endothelin (ET‐1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I‐ET‐1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET‐1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide‐binding proteins.

Functional endothelin-1 receptors in rat astrocytoma C6.

Rat astrocytoma C6 cells have been recently identified as target cells for ET-1, which stimulates inositol lipid turnover in these cells. It is shown here that binding of ET-1 to high-affinity receptors on C6 cells leads to 40-45% inhibition of isoproterenol-induced intracellular cyclic AMP accumulation, as well as to stimulation of inositol lipid turnover, both effects characterized by an absolute requirement of extracellular calcium. Moreover, ET-1, which has been generally reported to have a mitogenic effect on a variety of target cells including primary rat astrocytes, is shown here to stimulate or, alternatively, inhibit DNA synthesis in C6 cells, depending on the subclone considered.

Immortalization of brain capillary endothelial cells with maintenance of structural characteristics of the blood-brain barrier endothelium

SummaryEarly passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation, and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme andGriffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover, SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functionalβ1- andβ2-adrenergic receptors, as observed on isolated bovine brain capillaries.

Antibodies raised against β-adrenergic receptors stimulate adenylate cyclase

Abstract Antibodies raised in mice against β-adrenergic receptors purified from turkey erythrocyte membranes, specifically bind to cells which possess a β-adrenergic receptor and immunoprecipitate radiolabelled purified receptor. These antibodies stimulate the adenylate cyclase activity of the turkey erythrocytes, although they do not compete with the catecholamine hormones for binding to the β-adrenergic receptor. Thus the receptor-antibody interaction, although occuring at another site than the receptor-hormone interaction, may still trigger the enzymatic activity.

Towards the chemical and functional characterization of the β-adrenergic receptor

The biochemical characterization of the catecholamine β-adrenergic receptor from turkey erythrocyte membranes is rapidly progressing. The complex relationship with the adenylate cyclase and other membrane components which intervene in the hormonal stimulation of the cell is discussed in view of the effect of various ligands on the membrane-bound and affinity-purified receptor.

Biochemical and immunochemical analysis of β-adrenergic receptor adenylate cyclase complexes

Recent years have seen an exponential growth of reports concerning the biochemical analysis of hormone and neurotransmitter receptor-effecter systems*. The studies of the various subunits of the ~etylc~li~ receptor have reached the decisive stage 01 amino acid sequence determinations as well as morphological characterization by electron microscopy. Progress has not been so rapid for the &utrenergic receptor Icyclam 51 stem. mainly because of the lack of tissue equivalent to the electric organ of tbe Torpedo tish, which contains up to tOO&Ot_t :eceptor molecules per square micron. In their search for a favorable cell, specialists 5tfthe /3-adrenergic receptors have screened animals as diverse as the frog, turkey. mouse and man, and have studied estab fished cell lines as well as fmshly recovered tissues. Despite these difiiculties. the annlysis of litc catecholamine-sensitive adenylate cyc1as.z systems has continued to attract e~rmous attention mainly because of the crucial role of catechotar ant. in human physiology attested by the avaihbility of a large variety of agonist and antagonist compounds. An equally important reason for the interest in the ~-~nergic complex is the op~~unity to study in the same system an external hormonal signal and the transmission to the inside of the cell through activation of adenylate cyclase’-‘. In this review we will summarize the latest developments in the characterization and purification of the various comprments of the ~-~~nerg~ receptor-eyclase sys tern and will present the features of the syr+ tern for which a consensus has been reached by the major investigators in the geld.

Comparison of binding characteristics of endothelin receptors on subpopulations of astrocytes

Astrocytes in primary culture originating from different brain areas of the mouse embryo (striatum, cerebral cortex and mesencephalon) were compared for their [125I]-Endothelin-1 (ET-1) binding characteristics, in terms of affinity, binding capacity and specificity. Our results indicate that astrocytes from mesencephalon express about twice as many receptors as astrocytes from striatum or cortex (149,000 +/- 9700 vs 63,700 +/- 5600 and 81,900 +/- 5300, respectively), with similar affinities. Specificity patterns for the various peptides of the endothelins/sarafotoxins family (ET-1, -2, -3; SRTXa, b, c) are comparable in the three subpopulations of astrocytes: ET-1, -2 and SRTXb exhibit higher affinities than SRTXa and SRTXc. In addition, ET-3 and SRTXc seem to discriminate between different subsets of [125I]-ET-1 binding sites in the three subpopulations.

cAMP-dependent down-regulation of endothelin-1 receptors on rat astrocytoma C6 cells

The density of endothelin-1 (ET-1) receptors on rat astrocytoma C6 cells is down-regulated by activation of protein kinase C (PKC). We have investigated whether intracellular accumulation of cyclic adenosine monophosphate (cAMP) may also modulate surface ET-1 receptor number. The density of ET-1 receptors was measured by binding of [125I]ET-1 on rat astrocytoma C6 intact cells exposed to catecholamines, dibutyryl-cAMP or forskolin. Prolonged exposure of the cells to the beta-adrenergic agonists, isoproterenol or noradrenaline, results in a time- and dose-dependent decrease in cell surface ET-1 receptor number. This decrease proceeds slowly: maximal down-regulation is obtained by 6-7 h and sustained for up to 24 h in the presence of 10 microM isoproterenol. Since this down-regulation is mimicked by dibutyryl-cAMP (4 microM) and by forskolin (10 microM), we conclude that ET-1 receptors are susceptible to down-regulation through a cAMP-dependent pathway.

Altered binding properties of β-adrenergic receptors and lack of coupling to adenylate cyclase in P815 mastocytoma cells

P815, a murine mastocytoma cell line, possesses beta-adrenergic binding sites as assessed by using [3H]dihydroalprenolol (antagonist) and [3H]hydroxybenzylisoproterenol (agonist). The number of binding sites per cell was 29 000 for the agonist and 75 000 for the antagonist, as determined by direct binding assays and inhibition experiments on intact cells. On membrane preparations from the same cells, binding of alprenolol was only displaceable by antagonists, while stereospecific binding of hydroxybenzylisoproterenol was only displaceable by agonists. The P815 membranes also possessed an adenylate cyclase stimulated by Gpp(NH)p and NaF but not by 1-isoproterenol. The intracellular cAMP level of intact cells was not modulated by 1-isoproterenol or by 1-epinephrine, but was increased by forskolin. These results suggest that the beta-adrenergic receptor of P815 mastocytoma cells is non-functional. This may explain the failure of agonists to stimulate adenylate cyclase activity in these cells.

Assimilation of Ammonia During Sporogenesis of Saccharomyces cerevisiae: Effect of Ammonia and Glutamine

Summary: Comparison of the pools of glutamic acid and glutamine and of the specific activities of glutamine synthetase and glutamate dehydrogenases in sporulating a/α and non-sporulating α/α cells of Saccharomyces cerevisiae revealed a difference in their nitrogen metabolism. Glutamine synthetase and glutamine appeared to be necessary for the sporulation process, glutamine playing, at least, a catabolic role. However, exogenous glutamine as well as ammonia inhibited sporulation while glutamic acid did not. Glutamine seemed to act through its amino group. Both inhibitors had at least two sites of action, one effective early in sporulation and related to DNA synthesis and the other acting later and not related to it.