[A clinical case of depression finally explained by biology]. / Cas clinique. Un état dépressif finalement expliqué par la biologie.

We report the clinical case of a patient, 36 years old, presenting a major depressive disorder for more than 6 months. Somatic complaints (diarrhea and eczema) are also present. Thanks to a biological assessment, the diagnosis of celiac disease will be made and a strict gluten-free diet will be prescribed to the patient. This will allow the improvement of depressive symptoms and somatic complaints. This clinical case underlines the importance of the search for somatic causes in the development of a depressive symptomatology.

Nous rapportons le cas clinique d'une patiente, âgée de 36 ans, présentant un trouble dépressif majeur depuis plus de 6 mois. Des plaintes somatiques (diarrhée et eczéma) sont également présentes. Grâce à un bilan biologique, le diagnostic de maladie cœliaque sera posé et un régime strict sans gluten sera prescrit à la patiente. Celui-ci permettra l'amélioration de la symptomatologie dépressive et des plaintes somatiques. Ce cas clinique illustre l'importance de la recherche de causes somatiques dans la mise au point d'une symptomatologie dépressive.

Cystinyl and pyroglutamyl-beta-naphthylamide hydrolyzing activities are modified coordinately between hypothalamus, liver and plasma depending on the thyroid status of adult male rats.

The hypothalamus determinates metabolic processes in liver through endocrine and autonomic control. Hypothalamic neuropeptides, such as thyrotropin releasing hormone or vasopressin, have been involved in liver metabolism. The thyroid status influences metabolic processes including liver metabolism in modulating those hypothalamic peptides whose functional status is regulated in part by aminopeptidase activities. In order to obtain data for a possible coordinated interaction between hypothalamus, plasma and liver, of some aminopeptidase activities that may partially reflect the hydrolysis of those peptides, pyroglutamyl- (pGluAP) and cystinyl- (CysAP) beta-naphthylamide hydrolyzing activities were determined fluorimetrically, both in their soluble and membrane-bound forms, in eu- hypo- and hyperthyroid adult male rats. Hyperthyroidism and hypothyroidism were induced with daily subcutaneous injections of tetraiodothyronine (300 µg/kg/day) or with 0.03% methimazole in drinking water for 6 weeks. Results demonstrated significant changes depending on the type of enzyme and the thyroid status. The most striking changes were observed for CysAP in liver where it was reduced in hypothyroidism and increased in hyperthyroidism. Significant intra- and inter-tissue correlations were observed. While there were positive inter-tissue correlations between liver, plasma and hypothalamus in eu-and hypothyroid rats, a negative correlation between hypothalamus and liver was observed in hyperthyroidism. These results suggest the influence of thyroid hormones and an interactive role for these activities in the control of liver metabolism. The present data also suggest a role for CysAP and pGluAP activities in liver function linked to their activities in hypothalamus.

[Paradigm shift in the legislative approach of vulnerable people in Belgium]. / Changement de paradigme dans l'approche législative des personnes vulnérables en Belgique.

The application since September 2014 of the new 17 March 2013 law «reforming disability schemes and introducing a new protection status in accordance with human dignity¼, restates the legal approach to helping vulnerable people. The changes are complex and wide-ranging. This article describes the key elements of the reform, focusing particularly on the role of the medical doctor.

L'entrée en vigueur, début septembre 2014, de la loi du 17 mars 2013 «réformant les régimes d'incapacité et instaurant un nouveau statut de protection conforme à la dignité humaine¼, reformule l'approche juridique de l'aide aux personnes vulnérables. Les changements introduits sont complexes et de grande ampleur. Le présent article expose les éléments clés de la réforme, en se focalisant plus particulièrement sur le rôle du médecin.

Deletion of insulin-regulated aminopeptidase in mice decreases susceptibility to pentylenetetrazol-induced generalized seizures.

The peptide angiotensin IV (Ang IV) influences seizure susceptibility in rat and mouse models. Indeed, Ang IV has been shown to protect rats from limbic seizures in the focal pilocarpine model. Moreover, both anticonvulsive and antiepileptogenic effects of Ang IV have been reported in the acute pentylenetetrazol (PTZ) and kindling model of generalized seizures in mice. It has been hypothesized that the latter effects on seizures could be established via a modulatory effect on dopamine receptors in the basal ganglia or via an indirect interaction between Ang IV and adenosine A1 receptors. However, a possible role for insulin-regulated aminopeptidase (IRAP), the high affinity binding site for Ang IV, has not been studied yet. To unequivocally unravel the involvement of IRAP in generalized seizure generation, we investigated the susceptibility of male IRAP wild-type (IRAP(+/+)) and knock-out (IRAP(-/-)) mice to PTZ-induced seizures. Challenging these mice intravenously with PTZ resulted in significantly increased thresholds for myoclonic twitch and generalized clonic seizures with loss of righting reflexes in IRAP(-/-) mice compared to their IRAP(+/+) littermates. These behavioural data were confirmed by video-electrocorticography monitoring. Our study shows that IRAP(-/-) mice are less sensitive to the development of PTZ-induced seizures and suggests that IRAP is involved in generalized seizure generation.

Translocation of the insulin-regulated aminopeptidase to the cell surface: detection by radioligand binding.

BACKGROUND AND PURPOSE: Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes. EXPERIMENTAL APPROACH: After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H). KEY RESULTS: We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface. CONCLUSIONS AND IMPLICATIONS: Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.

Evaluation of bioactive saponins and triterpenoidal aglycons for their binding properties on human endothelin ETA and angiotensin AT1 receptors.

Different types of triterpenes including saponins and aglycons were evaluated for their ability to inhibit [3H] BQ-123 and [3H] angiotensin II binding to the human endothelin 1 ETA and angiotensin II AT1 receptors, respectively. Selectivity for only one of the two receptors was exhibited by asiatic acid and its saponins (ETA) and oleanolic acid (AT1). To a lesser extent betulinic acid, beta-amyrin and friedelin also showed selectivity for the ETA receptor. To address the question whether the effect of saponins on cell membranes might interfere with the normal binding of specific radioligands to their receptors, the activity of saponins with different haemolytic properties were compared. Highly haemolytic saponins such as alpha-hederin and beta-escine showed partial (60%) inhibition of radioligand-binding to the ETA receptor and complete inhibition (100%) to the AT1 receptor. Moreover, the haemolytically inactive kryptoescine, at the same concentration, caused complete inhibiton of radioligand-binding to both receptors, indicating that inhibition of receptor binding was not related to the membrane-interacting properties of saponins.

AT1 receptor antagonists.

Type 1 receptors (AT1) for the peptide hormone angiotensin II play a crucial role in the cardiovascular homeostasis. In this regard, several selective, orally active non-peptide antagonists have been developed for the treatment of hypertension and congestive heart failure. Pre-clinically, they have been routinely tested for their ability to inhibit angiotensin II induced contraction of rabbit aorta strips. This led to the distinction between surmountable antagonists, which only produce a parallel rightward shift of the angiotensin II concentration- induced response curve, and insurmountable antagonists that also decrease the maximal response. The molecular mechanism that is responsible for insurmountable antagonism has been extensively investigated in Chinese Hamster Ovary cells transfected expressing the human AT1 receptor. These experiments revealed that all biphenyltetrazole-countering AT1 receptor antagonists are competitive with angiotensin II and that the insurmountable behaviour of some of them is related to the formation of a long lasting/tight binding state of the antagonist-receptor complex. This may contribute to their long lasting clinical effect. This paper also focuses on the influence of a number of methodological approaches used to study AT1 receptor antagonists on their observed in vitro receptor binding properties.

Glucocorticoid receptor antagonists: new tools to investigate disorders characterized by cortisol hypersecretion.

Increased cortisol levels have been observed in patients suffering from a number of metabolic and psychiatric disorders. In some of these disorders a causal relationship has been suggested between the increased cortisol secretion and the observed clinical phenomena. Glucocorticoid receptor antagonists which block cortisol effects might have a benefit in both the diagnosis and treatment of these disorders. Selective glucocorticoid receptor antagonists with in vivo potency have not been described thus far, partly due to the similarity between the glucocorticoid and progesterone receptors. In the present studies, we report on three different chemical classes derived from the glucocorticoid/progestagen antagonist RU486. Selected compounds from the classes 11-monoaryl steroids, 11,21-bisaryl steroids and 11-aryl, 16-hydroxy steroids proved to be selective glucocorticoid receptor binders with in vivo antagonistic activity. Most compounds were able to pass the blood-brain barrier. These compounds offer the opportunity to investigate and possibly treat patients with a disturbed hypothalamus-pituitary-adrenal axis without side effects caused by an antiprogestagenic action.

New insights in insurmountable antagonism.

Antagonists that produce parallel rightward shifts of agonist dose-response curves with no alteration of the maximal response are traditionally classified as surmountable, while insurmountable antagonists also depress the maximal response. Although the longevity of the antagonist-receptor complex is quoted in many studies to explain insurmountable antagonism, slowly interconverting receptor conformations, allosteric binding sites, and receptor internalization have been evoked as alternative explanations. To complicate matters even further, insurmountable antagonism is not only drug-related; it may also depend on the tissue, species and experimental design. For the sake of drug development, it is important to elucidate the molecular mechanisms of insurmountable antagonism. New experimental approaches, such as intact cell studies and the use of computer-assisted simulations based on dynamic receptor models, herald the advent of better insight in the future.

Effects of BIBP3226 and BIBP3435 on cytosolic calcium in neuropeptide Y Y1 receptor-transfected Chinese hamster ovary cells and wild type CHO-K1 cells.

The NPY Y1-receptor selective antagonist BIBP3226 exerts a dual control on the cytosolic free calcium concentration ([Ca2+]i) in NPY Y1 receptor-transfected Chinese Hamster Ovary Cells (CHO-Y1 cells). It is a potent inhibitor of the NPY-evoked increase in [Ca2+]i. This can be ascribed to its antagonistic properties for the NPY Y, receptor since its less active stereoisomer, BIBP3435, is much less potent. However, when its concentration exceeds 1 microM, BIBP3226 produces a large increase in [Ca2+]i on its own. This effect is mimicked by BIBP3435 and it also occurs in wild type CHO-K1 cells. These latter cells do not contain high affinity binding sites for [3H]NPY and [3H]BIBP3226 and, hence, no endogenous NPY Y1 receptors. It is concluded that, at moderately high concentrations, the NPY Y1 receptor antagonist BIBP3226 and its entantiomer BIBP3435 are able to increase the [Ca2+ ]i in CHO cells either by stimulating another receptor or by directly affecting cellular mechanisms that are involved in calcium homeostasis.

Tight binding of the angiotensin AT(1) receptor antagonist.

Angiotensin II induces angiotensin AT(1) receptor internalization via Clathrin coated pits formation. We investigated whether insurmountable inhibition by the non-peptide antagonist 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl]-1H-benzimidazoline-7-carboxylic acid (candesartan) was related to receptor internalization. Mild acid treatment can discriminate between internalized and cell surface bound [(3)H]angiotensin II. In contrast, it provides no information about the subcellular localization of bound [(3)H]candesartan since this binding is acid resistant. The internalization of [(3)H]angiotensin II is rapidly inhibited in the presence of 0.4 M sucrose. Yet, no such rapid effect was noticed for [(3)H]candesartan. [(3)H]candesartan displays insurmountable/long lasting binding to the vast majority of both wild type and L(314) truncated rat angiotensin AT(1A) receptors with impaired receptor internalization. In agreement with previously published AT(1) angiotensin receptor visualization experiments, the present data suggest that non-peptide antagonist-angiotensin AT(1) receptor complexes remain at the cell surface. Insurmountable antagonism of candesartan is therefore independent from receptor internalization via clathrin-coated pits.

Biological screening of selected medicinal Panamanian plants by radioligand-binding techniques.

Nineteen plants from the Republic of Panama were selected by their traditional uses in the treatment of hypertension, cardiovascular, mental and feeding disorders and 149 extracts were screened using radioligand-receptor-binding assays. The methanol:dicloromethane extracts of the bark and leaves of Anacardium occidentale L., the leaves of Begonia urophylla Hook., the roots of Bocconia frutescens L., the stems and leaves of Cecropia cf.obtusifolia Bertol., the branches of Clusia coclensis Standl., the bark of Cochlospermum vitifolium (Willd.)Spreng., the roots of Dimerocostus strobilaceus Kuntze, the bark of Guazuma ulmifolia Lam., the leaves of Persea americana Mill. and the branches of Witheringia solanaceae L'Her. inhibited the [3H]-AT II binding (angiotensin II AT1 receptor) more than 50%. Only extracts of the roots of Dimerocostus strobilaceus Kuntze and the stems of Psychotria elata (Sw.) Hammel were potent inhibitors of the [3H] NPY binding (neuropeptide Y Y1 receptor) more than 50% and the ethanolic extracts of the leaves of Cecropia cf. obtusifolia Bertol., the leaves of Hedyosmum bonplandianum H.B.K., the roots of Bocconia frutescens L., the stem of Cecropia cf. obtusifolia Bertol. and the branches of Psychotria elata (Sw.) Hammel showed high inhibition of the [3H] BQ-123 binding (endothelin-1 ET(A) receptor) in a preliminary screening. These results promote the further investigation of these plants using the same assays.

A two-state receptor model for the interaction between angiotensin II type 1 receptors and non-peptide antagonists.

The interaction between non-peptide antagonists and the human angiotensin II type 1 (AT1) receptor in CHO-K1 cells was investigated by incubating the cells with antagonist, followed by a brief exposure to angiotensin II and measurement of the resulting inositol phosphate accumulation. The experimental data, expressed either as angiotensin II concentration-response curves or as antagonist concentration-inhibition curves, were in good agreement with computer-generated data according to a single-state model for the surmountable antagonist losartan and according to a two-step, two-state receptor model for the insurmountable antagonists candesartan, EXP3174, and irbesartan. Experimental and computer-generated data concerning the simultaneous exposure of the receptors to EXP3174 and losartan indicated that losartan produced a concentration-dependent restoration of the maximal response (angiotensin II concentration-response curves) as well as a rightward shift of the insurmountable portion of the EXP3174 inhibition curves, thus counteracting the higher-affinity EXP3174 binding. In conclusion, these findings provide further support for the concept that insurmountable and surmountable AT1 antagonists are mutually competitive and that insurmountable antagonist-receptor complexes may adopt different states.

Effect of maturation and aging on beta-adrenergic signal transduction in rat kidney and liver.

The characteristics of the beta-adrenergic signal transduction system were analyzed in kidney and liver membrane preparations from neonatal (2-3 days), mature (2 months), and old (2 years) rats. When comparing kidneys from adult to neonatal rats, we found a higher beta-receptor density and a higher percentage of beta(1)-receptor subtype, lower immunoreactive G(salpha)-protein, a lower ratio between the high and low molecular weight splice variant of G(salpha), lower immunoreactive G(ialpha)-protein, and lower basal adenylate cyclase activity. When comparing livers from adult to neonatal rats, we found lower beta-receptor density and basal adenylate cyclase activity. Very few differences could be detected when comparing mature to old kidneys or livers. Stimulated adenosine 3',5'-cyclic monophosphate (cAMP) synthesis was tissue- and age-dependent. In liver, G-protein- and beta-receptor-stimulated cAMP synthesis mirrored basal adenylate cyclase activity and was highest in liver from neonatal animals. In contrast, cAMP synthesis was significantly more stimulated in kidneys from mature animals than from neonatal and senescent rats. We conclude that: (i) the stoichiometry of the components within the beta-receptor/G-protein/adenylate cyclase complex is not fixed but is both tissue- and age-dependent; (ii) adenylate cyclase enzyme activity is possibly but not necessarily the rate-limiting step in the beta-receptor-mediated synthesis of cAMP; and (iii) there is in vivo evidence for a preferential co-expression of the large splice variant of the G(s)-protein and beta(2)-receptor subtype. It is speculated that this could have important physiological consequences for the development of the kidney.

Angiotensin II type 1 receptor antagonists. Why do some of them produce insurmountable inhibition?

Chinese hamster ovary (CHO) cells expressing human recombinant angiotensin II type 1 (AT(1)) receptors offer a useful experimental system in which antagonist binding and inhibition of AT-induced inositol mono-, bis-, and trisphosphate accumulation can be measured under identical experimental conditions. The major conclusions of the current work are: All investigated AT(1) antagonists are competitive with respect to AT. They bind to a common or overlapping binding site on the receptor in a mutually exclusive way. Reduction of the maximal angiotensin II response, i.e. insurmountable inhibition, is observed only when the cells are preincubated with candesartan, EXP3174, or irbesartan and is strictly related to the dissociation rate of the antagonist-receptor complex. On the other hand, inhibition by losartan is fully surmountable by AT, and its dissociation is very rapid. With respect to the binding kinetics, the antagonist-receptor complex can adopt a fast and a slow reversible state. The equilibrium between both states, which is dependent upon the nature of the antagonists, determines the extent of insurmountable inhibition. Consequently, the dissociation rate of the different antagonists correlates with the amount of insurmountable inhibition. In addition to the relatively slow dissociation of candesartan, reassociation to the receptor, which is measurable in CHO-AT(1) cells, likely contributes to its long-lasting blood pressure lowering effect in vivo.

Reversible and syntopic interaction between angiotensin receptor antagonists on Chinese hamster ovary cells expressing human angiotensin II type 1 receptors.

Evidence for a competitive type of interaction between angiotensin II type 1 (AT(1)) antagonists on Chinese hamster ovary cells expressing the human AT(1) receptor (CHO-AT(1)) was obtained by analyzing the binding of [(3)H]-2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H- ben zimidazoline-7-carboxylic acid ([(3)H]candesartan) and by measuring the AT-induced production of inositol phosphates. The AT(1) antagonists candesartan, 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]+ ++imid azole-5-carboxylic acid (EXP3174), or 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl- 4-yl)methyl]imidazole (losartan) produced a concentration-dependent increase in the apparent K(d) values of [(3)H]candesartan in saturation binding experiments, while the B(max) values were unchanged. Furthermore, the dissociation rate of the radioligand initiated by 1 microM unlabelled candesartan was not changed in the presence of 10 microM losartan, 10 microM EXP3174, or 10 microM irbesartan (2-n-butyl-4-spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)b iph enyl-4-yl) methyl]2-imidazolin-5-one)). Preincubation of the CHO-AT(1) cells with candesartan, EXP3174, and irbesartan caused a reduction in the maximal AT-induced inositol mono-, bis-, and trisphosphate production. This insurmountable effect was reversed in the presence of 1 microM losartan. In line with this finding, the insurmountable antagonist concentration-inhibition curves at 10 microM AT were shifted to the right in the presence of losartan. For candesartan this effect was concentration-dependent, yielding a pK(B) value for losartan of 7.7, which is similar to the pK(B) from previously obtained AT concentration-response curves. Finally, the dissociation rate of candesartan, EXP3174, irbesartan, and losartan was determined by measuring the recovery of AT responses after antagonist pretreatment and washing of the cells with medium containing 1 microM losartan to prevent re-association of the insurmountable antagonists. In addition, similar kinetic data were obtained from the slowing of the [(3)H]candesartan association rate to antagonist preincubated cells.

Interaction between the partially insurmountable antagonist valsartan and human recombinant angiotensin II type 1 receptors.

The interaction between the AT1 receptor-selective antagonist valsartan, and its human receptor, was investigated by direct radioligand binding as well as by its inhibition of angiotensin II induced inositol phosphate accumulation in CHO cells expressing human recombinant AT1 receptors. Specific binding of [3H]-valsartan rapidly reached equilibrium at 37 degrees C. It was saturable and occurred to a homogeneous class of sites with a K(D) of 0.88+/-0.076. It was inhibited by other AT1 receptor antagonists with the same potency order as previously described for the binding of [3H]-angiotensin II and [3H]-candesartan to human AT1 receptors (i.e. candesartan > or = EXP3174 > valsartan = irbesartan = angiotensin II > losartan). When valsartan and angiotensin II were applied simultaneously to the CHO-AT1 cells. the antagonist caused a rightward shift of the angiotensin II concentration response curve. Hence, valsartan interacts with the AT1 receptor in a manner that is competitive with angiotensin II. Pre-incubation of the cells with 0.5, 5 and 50 nM valsartan caused an additional, concentration-dependent, up to 55% decline of the maximal response. The partial nature of this insurmountable inhibition by valsartan was confirmed by biphasic antagonist concentration-inhibition curves. These data reflect the co-existence of a fast reversible/surmountable as well as a tight binding/insurmountable valsartan receptor complex. In agreement, pre-incubation of the CHO-AT1 cells with 5 and 50 nM valsartan produced a partial inhibition of the angiotensin II induced increase of the free intracellular calcium concentration. [3H]-Valsartan dissociated from its receptors with a half-life of 17 min. In functional recovery experiments with valsartan-pre-treated cells, the angiotensin II-mediated response was half-maximally restored within approximately 30 min. These kinetic data suggest that the insurmountable inhibition by valsartan is related to its relatively slow dissociation from the human AT1 receptors.

Lys(199) mutation of the human angiotensin type 1 receptor differentially affects the binding of surmountable and insurmountable non-peptide antagonists.

Many slow dissociating (insurmountable) non-peptide angiotensin type 1 receptor (AT1) antagonists contain,besides the acidic biphenyltetrazole substructure of losartan, a second acidic group to stabilise antagonist-receptor complexes. To investigate the involved basic amino-acids of the human AT1-receptor, wild-type and mutant receptors were transiently transfected in CHO-K1 cells and characterised by [3H]candesartan binding. Lys199-->Gln substitution decreased the affinity 45-fold for candesartan (95% insurmountable),18-fold for EXP3174 (70% insurmountable), 10-fold for irbesartan (40% insurmountable) and 5-fold for losartan (surmountable). His256 -->Ala substitution had only minor effects. This suggests that Lys199 is important for the tight binding of non-peptide antagonists.