Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function.

1 Previous studies in our laboratory have shown that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE-4) inhibitor, is a potent activator of the hypothalamo-pituitaryadrenal (HPA) axis when given orally or intraperitoneally (i.p.) to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro methods were used to compare the e ects of denbufylline on HPA function with those of two other selective PDE-4 inhibitors, rolipram and BRL 61063 (1,3-dicyclopropylmethyl-8-amino-xanthine 71 , i.c.v.) was also ine ective in this regard although at a higher dose (1 mg kg 71 , i.c.v.) it produced a small but signi®cant (P50.05) increase in ircorticosterone release. Denbufylline also increased the serum ir-LH concentration when given peripherally (0.2 ± 0.6 mg kg 71 , i.p., P50.05) or centrally (100 ng kg 71 , i.c.v., P50.05) but rolipram (1.6 ± 200 mg kg 71 , i.p. or 8 ng ± 1 mg kg 71 , i.c.v.) and BRL 61063 (0.25 ± 30 mg kg 71 , i.p. or 1 ng ± 1 mg kg 71 , i.c.v.) did not (P40.05). 3 In vitro rolipram (10 mM, P50.01), denbufylline (1 mM, P50.001) and BRL 61063 (1 and 10 mM, P50.05) stimulated the release of corticotrophin releasing hormone (ir-CRH-41) but lower concentrations of the drugs were without e ect as also was BRL 61063 at 100 mM (P40.05); the rank order of potency was thus BRL 610634rolipram4denbufylline. The adenylyl cyclase activator forskolin (100 mM, P50.01) also stimulated the release of ir-CRH-41, producing e ects which were additive with those of rolipram and denbufylline but not with those of BRL 61063. The secretory responses to forskolin (100 mM) were accompanied by a highly signi®cant increase in the cyclic AMP content of the hypothalamic tissue (P50.005). Rolipram (10 mM) also signi®cantly (P50.05) elevated the hypothalamic cyclic AMP but denbufylline (10 mM) and BRL 61063 (10 mM) did not. However, all three PDEinhibitors potentiated the rise in cyclic AMP induced by forskolin (P50.05). None of the drugs tested, alone or in combination, modi®ed the release of arginine vasopressin (ir-AVP) from the hypothalamus. 4 Rolipram (100 mM), denbufylline (100 mM) and BRL 61063 (100 mM) stimulated the release of corticotrophin (ir-ACTH) from pituitary tissue in vitro (P50.05) but in lower concentrations they were without signi®cant e ect. In addition, rolipram (10 mM, P50.05), denbufylline (0.1 mM, P50.05) and BRL 61063 (10 mM, P50.05) potentiated the signi®cant (P50.05) rises in ir-ACTH secretion induced by forskolin (100 mM). Forskolin (100 mM) also produced a highly signi®cant increase (P50.01) in the tissue cyclic AMP content which was further potentiated by rolipram (10 mM), denbufylline (10 mM) and BRL 61063 (10 mM) which, alone did not a ect the cyclic AMP content of the tissue. 5 Since both denbufylline and BRL 61063 possess signi®cant adenosine A 1 receptor blocking activity, further studies examined the potential in¯uence of these receptors on the secretion in vitro of CRH-41, AVP and ACTH. The release of ir-CR...

Preliminary studies in our laboratories showed that the synthetic xanthine analogue denbufylline, a selective type 4 phosphodiesterase (PDE‐4) inhibitor, is a potent activator of the hypothalamo‐pituitary‐adrenal (HPA) axis when given orally to adult male rats. This paper describes the results of experiments in which well established in vivo and in vitro models were used to (a) examine further the effects of denbufylline on HPA function and (b) identify the site and mode of action of the drug within the axis. In vivo, administration of denbufylline (0.1–2.5 mg kg−1, i.p.) produced a significant increase in the serum corticosterone concentration; maximal responses were attained at a dose of 1.0 mg kg−1 (P < 0.01 vs. vehicle control, Scheffe's test). However, when denbufylline was administered by intracerebroven‐tricular injection (0.05‐1 μg kg−1) it failed to influence significantly the serum corticosterone concentration (P > 0.05 vs. vehicle control, Scheffe's test). The adrenocortical responses to peripheral injections of denbufylline (1 mg kg−1, i.p.) were reduced in rats in which the secretion of endogenous corticotrophin releasing factors (CRFs) from the hypothalamus was blocked pharmacologically (P < 0.01 vs. controls, Scheffe's test). However, denbufylline (0.1 mg kg−1, i.p.) potentiated the significant (P < 0.01) increases in serum corticosterone concentration provoked in ‘CRF blocked rats’ by hypothalamic extract (5 hypothalamic extracts kg−1, i.v.) although it failed to influence (P > 0.05) the relatively moderate increases in corticosterone secretion evoked by CRH‐41 (2 mg kg−1, i.v.). In vitro, denbufylline (0.01‐1 mM) evoked small but significant (P < 0.05) increases in the release of ACTH from rat anterior pituitary segments; furthermore, at these and lower concentrations (0.01 μm‐1 mM), it potentiated the adrenocorticotrophic responses to sub‐maximal concentrations of hypothalamic extract (P < 0.01) and forskolin (0.1 mM, P < 0.01) but not those to CRH‐41 (10 nM) or 8‐bromo‐cyclic AMP (1–100 μm). In addition, denbufylline (0.1 mM) increased the anterior pituitary cyclic AMP content (P < 0.05) and potentiated the rises in tissue content of the cyclic nucleotide induced by hypothalamic extract (0.1 hypothalamic equivalents ml−1, P < 0.01) and forskolin (0.1 mM, P < 0.01) but not by CRH‐41 (10 nM, P < 0.05). By contrast, denbufylline (1 μm‐1 mM) failed to influence the release of AVP from rat isolated hypothalami and stimulated the secretion of CRH‐41 (P < 0.01) release only at the highest concentration tested (1 mM). The results suggest that the stimulatory actions of denbufylline on the hypothalamo‐pituitary‐adrenocortical axis are exerted predominantly at the level of the anterior pituitary gland and that they may be attributed, at least in part, to inhibition of type 4 phosphodiesterase enzymes.

Epindolidione (H 2 L), ah eteroatom-modifieda nalogue of tetracene and as tructural isomer of indigo, forms dinuclear complexesw ith [RuX 2 ] 2 + ,X = bpy (2,2'-bipyridine, [1] 2 +)o rpap (2-phenylazopyridine, [2] 2 +), in its doubly deprotonatedb ridging form m-L 2À .The dications in compounds meso-[1](ClO 4) 2 and meso-[2](ClO 4) 2 ,[ X 2 Ru(m-L)RuX 2 ](ClO 4) 2 , contain five-membered chelate rings N-CÀC-O-Ru II with p bridged metalsa ta ni ntramolecular distance of 7.19 .S tepwise reversible oxidation and reduction is mainly ligand centered (oxidation:L 2À ;r eduction:X), as deduced from EPR of one-electron oxidized and reduced intermediates and from UV/Vis/NIR spectroelectrochemistry,s upported by TD-DFT calculation results.T he results for [1](ClO 4) 2 and [2](ClO 4) 2 are qualitatively similart ot he ones observed with the deprotonated indigo-bridged isomers with their six-membered chelate ring structures, confirming the suitability of both p systems for molecular electronicsa pplications, low-energy absorptions, and multiple electron transfers. Scheme1.Structuralisomers of indigo and epindolidione. Scheme2.The two diruthenium complexes [1](ClO 4) 2 and [2](ClO 4) 2 with doublydeprotonated epindolidione presented in this work.