Odile Kellermann

Cross-talk between 5-Hydroxytryptamine Receptors in a Serotonergic Cell Line INVOLVEMENT OF ARACHIDONIC ACID METABOLISM

The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. We took advantage of a serotonergic cell line to elucidate cross-talks between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed since day 2 of the serotonergic differentiation while 5-HT2A receptors are induced at day 4. We first established that 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and that the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation although a 5-HT2A/PLA2 positive coupling was evidenced. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis.

Functions of serotonin in hypoxic pulmonary vascular remodeling.

In lung vasculature, reversible constriction of smooth muscle cells exists in response to acute decrease in oxygen levels (hypoxia). Progressive and irreversible structural remodeling that reduces blood vessel lumen takes place in response to chronic hypoxia and results in pulmonary hypertension. Several studies have shown a role of serotonin in regulating acute and chronic hypoxic responses. In this review the contribution of serotonin, its receptors and transporter in lung hypoxic responses is discussed. Hypoxic conditions modify plasma levels of serotonin, serotonin transporter activity, and expression of 5-HT1B and 5-HT2B receptors. These appear to be required for pulmonary vascular cell proliferation, which depends on the ratio between reactive oxygen species and nitric oxide. A heterozygous mutation was identified in the 5-HT2B receptor gene of a patient who developed pulmonary hypertension after fenfluramines anorexigen treatment. This C-terminus truncated 5-HT2B mutant receptor presents lower nitric oxide coupling, and higher cell proliferation capacity than the wild-type receptor. Under low oxygen tension, cells increase the transcription of specific genes via stabilization of the transcription factor hypoxia-inducible factor (HIF)-1. Factors such as angiotensin II or thrombin that can also control HIF-1 pathway contribute to pulmonary vascular remodeling. The 5-HT2B receptor via phosphatidylinositol-3 kinase/Akt activates nuclear factor-κB, which is involved in the regulation of HIF-1 expression. A control of HIF-1 by 5-HT2B receptors explains why expression of pulmonary vascular remodeling factors, such as endothelin-1 or transforming growth factor-β, which is HIF-1-α regulated, is not modified in hypoxic 5-HT2B receptor mutant mice. Understanding the detailed mechanisms involved in lung hypoxic responses may provide general insight into pulmonary hypertension pathogenesis.

Sequential onset of three 5-HT receptors during the 5-hydroxytryptaminergic differentiation of the murine 1C11 cell line.

1 The murine 1C11 clone, which derives from a multipotential embryonal carcinoma cell line, has the features of a neuroectodermal precursor. When cultured in the presence of dibutyryl cyclic AMP, the 1C11 cells extend bipolar extensions and express neurone‐associated markers. After 4 days, the resulting cells have acquired the ability to synthesize, take up, store and catabolize 5‐hydroxytryptamine (5‐HT). We have thus investigated the presence of 5‐HT receptors during the 5‐hydroxytryptaminergic differentiation of this inducible 1C11 cell line. 2 As shown by the binding of [125I]‐GTI and the CGS 12066‐dependent inhibition of the forskolin‐induced cyclic AMP production, functional 5‐HT1B/1D receptors become expressed on day 2 of 1C11 cell differentiation. The density of these receptors remained unchanged until day 4. 3 The same holds true for the 5‐HT2B receptor, also identified by its pharmacological profile and its positive coupling to the phosphoinositide cascade. 4 On day 4 of 1C11 cell differentiation, a third 5‐HT receptor, pharmacologically and functionally similar to 5‐HT2A, had become induced. 5 Strikingly, the amounts of each transcript encoding 5‐HT1B, 5‐HT2A and 5‐HT2B receptor did not vary significantly during the time course of the 1C11 5‐hydroxytryptaminergic differentiation. 6 The clone 1C11 may thus provide a useful in vitro model for studying regulation(s) between multiple G‐linked receptors as well as the possible role of 5‐HT upon the expression of a complete 5‐hydroxytryptamine phenotype.

Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma‐derived neuroectodermic precursor cells

The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn‐finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over‐expressed in brain. At embryonic daysu200312 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic dayu200315, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post‐natal dayu200310, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb.