Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Proteolytic activity of proteasome on myofibrillar structures

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than -actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.     

Changes in organic solutes,volume, energy state,and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo¹H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.¹H-,¹³C-and³¹P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo³¹P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.To whom to address reprint requests.     

A new monoclonal antibody (3D3) generated with human respiratory mucins and directed against Lewis determinants

We have prepared a monoclonal antibody (MAb), 3D3, raised againstpurified human respiratory mucins. This antibody recognizedmucins and proteolytically derived glycopeptides. The epitoperecognized by the antibody was destroyed by -L-fucosidase, indicatingthat it was present on the carbohydrate moieties. Structuralspecificity was determined by adsorption on a variety of synthetic,insolubilized oligosaccharides. Several lines of evidence indicatethat the 3D3 MAb reacted strongly with the Lewis (Le^b) antigen,but also recognized Le^a and Le^y determinants. This antibodymight be useful to study mucin secretion. human bronchial mucins Lewis b     

Methyl-4 formyl-7 cyclopenta(c)pyranne isole apres hydrolyse acide de Viburnum tinus

Viburtinal (4-methyl-7-formylcyclopenta(c)pyrane), yet unknown, was obtained by acid hydrolysis of the esters extracted from Viburnum tinus. Its structure was deduced by spectroscopic methods.     

Potent and specific blockade by AH 22216 of histamine-H2-receptor-mediated acid secretion in isolated rabbit gastric cells

AH 22216 is a new histamine-H₂-receptor antagonist which possesses a triazole ring. When compared to cimetidine, AH 22216 is about 100 times more potent (Ki = 0.21×10^–8 M) in inhibiting histamine-stimulated acid secretion in isolated rabbit gastric cells. These two antihistamines have no effect on carbachol-stimulated acid secretion in the system. The data indicate that AH 22216 interacts directly and specifically on the gastric H₂-receptor of the parietal cell and are consistent with the reported pharmacological potencies of AH 22216 and cimetidine on histamine-induced gastric-acid secretion in vivo. AH 22216 could thus be a useful therapeutic agent in patients with peptic ulcers.     

v-myb Transformation of Xeroderma pigmentosum human fibroblasts: Overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum “normal” fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental “normal” AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotipically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

Detection of ginkgolide A in Ginkgo biloba cell cultures

Ginkgo biloba cells were cultured in two 500 mL shake flasks and in 2 L and 6 L immobilization bioreactors using MS medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30 g.L^–1 sucrose. Specific growth rates were 0.06 d^–1, 0.11 d^–1 and 0.07 d^–1 for the 2 L and 6 L bioreactors and shake flask cultures, respectively. Extracellular phosphate, nitrate, ammonium and carbohydrate uptake rates of the bio reactor cultures were approximately 17 to 39% slower than those of shake flask cultures. The specific oxygen uptake and carbon dioxide transfer rates of immobilized Ginkgo biloba cells ranged from 0.027 to 0.041 mmol O₂.g^–1.d.w.hr^–1 (maximum uptake at 14 days) and 0.020 to 0.057 mmol CO₂g. ^–1.d.w.hr^–1 (maximum production at 14 days). Extracts from the biomass of the two immobilized and shake flask suspension cultures were analysed for ginkgolide A by GC-MS. Yields of 7, 17, 19 and 7 ng.g. ^–1d.w. of ginkgolide A were determined for shake flask 1, shake flask 2 and the 2 L and 6 L immobilized cultures, respectively. Traces of ginkgolide B were detected with the signal to noise ratio, however, being too low for positive confirmation of this last product.Abbreviations CTR Carbon dioxide transfer rate - DO Dissolved oxygen - g.d.w. Gram dry weight - GA Ginkgolide A - GB Ginkgolide B - GC Gas chromatography - GC-MS Gas chromatography-mass spectrometry - HPLC High performance liquid chromatography - K Kinetin - MS Murashige and Skoog salt medium - N₁K₁MS Complete Murashige and Skoog medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30g.L^–1 sucrose - NAA Naphthaleneacetic acid - OTR Oxygen transfer rate - PAF Platelet Aggregating Factor - q_CO2 Specific carbon dioxide production rate - q_O2 Specific oxygen uptake rate - u Specific growth rate