3-[5-(4,5-dihydro-1H-imidazol-2-yl)-furan-2-yl]phenylamine (Amifuraline), a promising reversible and selective peripheral MAO-A inhibitor.

On the basis of the observation that the central side effects of MAO inhibitors may represent a major limit for their use in pathological processes involving peripheral MAOs, we investigated the possibility of generating novel inhibitors able to target specifically peripheral MAOs. To address this issue, we designed compounds 7-28. From biological results, the 2-(5-phenyl-furan-2-yl)-4,5-dihydro-1H-imidazole (Furaline, 17) proved to be a suitable lead. In fact, in enzyme assays on homogenate preparation from rat liver and HEK cells expressing MAO-A or MAO-B, compounds possessing the frame of 17 behaved as selective and reversible MAO-A inhibitors. Interestingly, in in vivo studies the amino derivative 21 (Amifuraline), endowed with good hydrophilic character, was able to significantly inhibit liver but not brain MAO-A.

Exogenous and fibroblast growth factor 2/epidermal growth factor-regulated endogenous cytokines regulate neural precursor cell growth and differentiation.

Neurospheres (NSs) are clonal cellular aggregates composed of neural stem cells and progenitors. A comprehensive description of their proliferation and differentiation regulation is an essential prerequisite for their use in biotherapies. Cytokines are essential molecules regulating cell precursor fate. Using a gene-array strategy, we conducted a descriptive and functional analysis of endogenous cytokines and receptors expressed by spinal cord-derived NSs during their growth or their differentiation into neuronal and glial cells. NSs were found to express approximately 100 receptor subunits and cytokine/secreted developmental factors. Several angiogenic factors and receptors that could mediate neural precursor cell-endothelial cell relationships were detected. Among them, receptor B for endothelins was highly expressed, and endothelins were found to increase NS growth. In contrast, NSs express receptors for ciliary neurotrophic factor (CNTF), bone morphogenetic protein (BMP), interferon (IFN)-gamma, or tumor necrosis factor (TNF)-alpha, which, when added in the growth phase, led to a dramatic growth reduction followed by a reduction or a loss of oligodendrocyte formation on differentiation. In addition, NSs synthesize fibroblast growth factor 2/epidermal growth factor (FGF2/EGF)-regulated endogenous cytokines that participate in their growth and differentiation. Notably, BMP-7 and CNTF were expressed during expansion, but upon differentiation there was a remarkable switch from BMP-7 to BMP-4 and -6 and a sharp increase of CNTF. Reintroduction of growth factors reverses the BMP expression profile, indicating growth factor-BMP cross-regulations. The role of endogenous CNTF was investigated by deriving NSs from CNTF knockout mice. These NSs have an increased growth rate associated with reduction of apoptosis and generate astrocytes with a reduced glial fibulary acidic protein (GFAP) content. These results demonstrate the combined role of endogenous and exogenous cytokines in neural precursor cell growth and differentiation.

Essential interaction of Egr-1 at an islet-specific response element for basal and gastrin-dependent glucagon gene transactivation in pancreatic alpha-cells.

The peptide hormone gastrin is secreted from G cells of the gastric antrum and is the main inducer of gastric acid secretion via activation of its receptor the cholecystokinin 2 (CCK2) receptor. Both gastrin and CCK2 receptors are also transiently detected in the fetal pancreas and believed to exert growth/differentiation effects during endocrine pancreatic development. We demonstrated previously that whereas gastrin expression is extinguished in adult pancreas, CCK2 receptors are present in human glucagon-producing cells where their activation stimulates glucagon secretion. Based on these findings, we investigate in the present study whether gastrin regulates glucagon gene expression. To this aim, the CCK2 receptor was stably expressed into a glucagon-producing pancreatic islet cell line, and a glucagon-reporter fusion gene was transiently transfected in this new cellular model. We report that gastrin stimulates glucagon gene expression in glucagon-producing pancreatic cells. By using progressively 5'-increased sequences of the glucagon gene, gastrin responsiveness was located within the minimal promoter. Moreover, we clearly identified early growth response protein 1 (Egr-1) as an essential transcription factor interacting with the islet cell-specific G4 element. Egr-1 was shown to be essential for basal and gastrin-dependent glucagon gene transactivation. Furthermore, our results demonstrate that the MEK1/ERK1/2 pathway couples the CCK2 receptor to nuclearization and DNA binding of Egr-1. In conclusion, our data provide new information concerning the transcriptional regulation of the glucagon gene. Moreover they open new working hypothesis with reference to a potential role of gastrin in glucagon-producing pancreatic cells.

Substrate-dependent regulation of MAO-A in rat mesangial cells: involvement of dopamine D2-like receptors.

In the present study, we investigated the existence of a back-regulation of the catecholamine-degrading enzyme monoamine oxidase (MAO)-A by dopamine in rat renal cells. In proximal tubule cells, MAO-A expression was not modified after dopamine receptor stimulation. In contrast, in mesangial cells, enzyme assay and Western blots showed that MAO activity and protein increased by approximately 80% after 48-h incubation with the D(2)-like receptor agonist bromocriptine and quinpirole but not with the D(1)-like receptor agonist SKF-38393. This effect was prevented by the D(2)-receptor antagonist sulpiride and domperidone. The increase in MAO-A protein was preceded by an augmentation of MAO-A mRNA that was prevented by the transcriptional inhibitor actinomycin D. Bromocriptine effect was mimicked by the PKA inhibitor H89 and inhibited by the PKA activator 8-bromo-cAMP. These results show for the first time the existence of a dopamine-dependent MAO-A regulation involving D(2)-like receptors, inhibition of the cAMP-PKA pathway, and an ex novo enzyme synthesis.

Dopamine D4 receptor expression in rat kidney: evidence for pre- and postjunctional localization.

Dopamine D4 receptors mediate inhibition of vasopressin-dependent sodium reabsorption by dopamine in collecting tubules. At present, the distribution of D4 receptors in other renal districts remains an open issue. The renal distribution of D4 receptor was assessed in normally innervated and denervated male Sprague-Dawley rats by quantitative immunohistochemistry using an anti-dopamine D4 receptor rabbit polyclonal antibody. D4 receptor protein immunoreactivity was observed perivascularly in the adventitia and the adventitia-media border. The density of perivascular dopamine D4 receptor was higher in afferent and efferent arterioles than in other segments of the renal vascular tree. Renal denervation abolished perivascular dopamine D4 receptor protein immunoreactivity. In renal tubules, the epithelium of collecting tubules showed the highest dopamine D4 receptor protein immunoreactivity, followed by the epithelium of proximal and distal tubules. No dopamine D4 receptor protein immunoreactivity was observed in the epithelium of the loop of Henle. Denervation did not change dopamine D4 receptor protein immunoreactivity in renal tubules. These results indicate that rat kidney expresses dopamine D4 receptors located both prejunctionally and nonprejunctionally in collecting, proximal, and distal tubules. This suggests that the dopamine D4 receptor may be involved in the control of neurotransmitter release and in renal hemodynamic and tubule function.

Genetic, pharmacological and functional analysis of cholecystokinin-1 and cholecystokinin-2 receptor polymorphism in type 2 diabetes and obese patients.

Cholecystokinin (CCK) and gastrin (G) and their receptors (CCK1 and CCK2) are involved in multiple physiological functions. Notably, CCK1R plays a role in the regulation of food intake whereas both CCK1R and CCK2R play a role in the regulation of pancreatic endocrine function. CCK1R and CCK2R may therefore serve as pharmacological targets in diabetes and obesity and genes encoding these receptors may be candidate genes in the pathogenesis of the diseases. In this study, we used single nucleotide polymorphism analysis and allele specific amplification for mutation screening of the CCK2 receptor gene and family linkage study. Mutated receptors were constructed, expressed in COS-7 cells for analysis of their binding and functional properties. V125I-CCK2 receptor variant was found in 2 out of 18 type 2 diabetes mellitus families tested. V125I mutation co-segregated in those 2 initial families, but further association studies showed that this mutation was not associated with diabetes or early age at diagnosis of the disease. V125I-CCK2 receptor high affinity sites exhibited a 2-fold enhanced binding affinity for CCK which was correlated to a slightly increased potency in coupling to inositol phosphate production. Since CCK2 receptor is expressed in pancreatic glucagon-producing cells in humans and is involved in secretion of glucagon, an increase of binding affinity of the mutated CCK2 receptor could enhance glucagon secretion in patients bearing V125I mutation. We also characterized a mutant of the CCK1 receptor which was previously identified in an obese patient. This mutant, V365I-CCK1, demonstrated a decreased level of expression (26%) and efficacy (25%) to stimulate inositol phosphates. It can therefore be expected that in humans bearing V365I mutation, decreases in CCK1 receptor expression and coupling efficiency may affect CCK-induced regulation of satiety. Polymorphism or mutations in the CCK receptors may be involved in type 2 diabetes mellitus and obesity. However, further studies are necessary to precisely evaluate this role in humans.