[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.

[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.

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.

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.

Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO-K1 cells expressing human angiotensin II AT1 receptors.

1. CHO-K1 cells that were stably transfected with the gene for the human AT1 receptor (CHO-AT1 cells) were used for pharmacological studies of non-peptide AT1 receptor antagonists. 2. In the presence of 10 mM LiCl, angiotensin II caused a concentration-dependent and long-lasting increase of inositol phosphates accumulation with an EC50 of 3.4 nM. No angiotensin II responses are seen in wild-type CHO-K1 cells. 3. [3H]-Angiotensin II bound to cell surface AT1 receptors (dissociates under mild acidic conditions) and is subject to rapid internalization. 4. Non-peptide selective AT1 antagonists inhibited the angiotensin II (0.1 microM) induced IP accumulation and the binding of [3H]-angiotensin II (1 nM) with the potency order: candesartan > EXP3174 > irbesartan > losartan. Their potencies are lower in the presence of bovine serum albumin. 5. Preincubation with the insurmountable antagonist candesartan decreased the maximal angiotensin II induced inositol phosphate accumulation up to 94% and, concomitantly, decreased the maximal binding capacity of the cell surface receptors. These inhibitory effects were half-maximal for 0.6 nM candesartan and were attenuated by simultaneous preincubation with 1 microM losartan indicating a syntopic action of both antagonists. 6. Losartan caused a parallel rightward shift of the angiotensin II concentration-response curves and did not affect the maximal binding capacity. EXP3174 (the active metabolite of losartan) and irbesartan showed a mixed-type behavior in both functional and binding studies. 7. Reversal of the inhibitory effect was slower for candesartan as compared with EXP3174 and irbesartan and it was almost instantaneous for losartan, suggesting that the insurmountable nature of selective AT1 receptor antagonists in functional studies was related to their long-lasting inhibition.

Different agonist binding properties of M1 and M2 muscarinic receptors in calf brain cortex membranes.

The muscarinic antagonist 1-[benzilic 4,4'-3H]-quinuclidinyl benzilate [3H]-QNB) bound to a single class of non-cooperative sites in calf cerebral cortex membranes (KD = 0.29 nM and Bmax = 1.06 pM/mg protein). Computer-assisted analysis of the shallow pirenzepine/[3H]-QNB competition binding curves indicated that 68% of these sites were of the M1-subtype and the remaining 32% of the M2 subtype. Respective Ki-values for pirenzepine were 27 nM and 1.14 microM. Binding characteristics of the antagonist atropine and of the agonist carbachol for M2 were evaluated by performing competition binding with 0.5 nM [3H]-QNB in the presence of 2 microM pirenzepine. The binding characteristics for the M1 receptors were obtained indirectly by subtracting the curve for M2 from the total curve, or directly by competition binding with 0.3 nM [3H]-pirenzepine. Atropine competition curves were steep for M1 and M2 and were not affected by 1 mM GTP nor by 1 mM N-ethylmaleimide. The carbachol competition curve was shallow for M2. The steep curves for M1 indicate that this receptor subclass was only composed of low agonist affinity sites. GTP, which caused a rightward shift and a steepening of the carbachol competition curve for M2, did not affect the curves for M1. N-ethylmaleimide provoked a leftward shift and a steepening of the carbachol competition curve for M2 and abolished GTP modulation. A leftward shift was also observed for M1, but of a smaller magnitude (i.e. 3-4-fold for M1 compared to 17-fold for M2). These data suggest that, in calf brain cortex, M1 and M2 receptors show different susceptibility towards GTP and N-ethylmaleimide modulation.

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.

Agonist mediated conformational changes of solubilized calf forebrain muscarinic acetylcholine receptors.

Muscarinic receptors in calf forebrain membranes can be identified by the specific binding of the radiolabelled antagonist [3H]dexetimide. These receptors (2.8 pM/mg protein) comprise two non-interconvertible subpopulations with respectively high and low agonist affinity but with the same antagonist affinity. For all the agonists tested the low affinity sites represent 85 +/- 5% of the total receptor population. 0.5% Digitonin solubilized extracts contain 0.8 pM muscarinic receptor/mg protein. In contrast with the membranes, these extracts contain only sites with low agonist affinity. The alkylating reagent N-ethylmaleimide causes an increase of the acetylcholine affinity for the low affinity sites in membranes as well as for the solubilized sites. This effect is time dependent until a maximal 3-fold increase in affinity is attained. The rate of N-ethylmaleimide action is enhanced by the concomitant presence of agonists. In contrast, N-ethylmaleimide does not affect antagonist binding. This suggests that agonists mediate a conformational change of both the membrane bound low affinity muscarinic sites and of the solubilized sites, resulting in their increased susceptibility towards NEM alkylation.

Radiolabelling of the human 5-HT2A receptor with an agonist, a partial agonist and an antagonist: effects on apparent agonist affinities.

Previous work has shown that 5-hydroxytryptamine (5-HT)2A receptors can be radiolabelled with various radioligands, including partial agonists, such as [125I]-DOI and [3H]-DOB, and antagonists, such as [3H]-ketanserin and [3H]-spiperone. Because 5-HT has high affinity for the 5-HT2A receptor when displacing [3H]-DOB, the purpose of the present study was to determine whether or not the receptor could be labelled with [3H]-5-HT and what would be the effect of labelling the receptor with various radioligands having differing efficacies at the receptor. Consequently, the human 5-HT2A receptor stably expressed in NIH 3T3 cells was radiolabelled with the endogenous agonist [3H]-5-HT, the partial agonist [3H]-DOB, and the antagonist [3H]-ketanserin. The receptor could be radiolabelled with [3H]-5-HT with a Kd value of 1.3 +/- 0.1 nM and a Bmax value of 3461 +/- 186 fmoles/mg protein and the radiolabelling was sensitive to the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate (GMP-PNP). Ketanserin labeled significantly more receptors (Kd = 1.1 +/- 0.1 nM: Bmax = 27,684 +/- 1500 fmoles/mg protein) than [3H]-DOB (Kd = 0.8 +/- 0.08 nM: Bmax = 8332 +/- 16 fmoles/mg protein) which, in turn, labelled significantly more receptors than [3H]-5-HT. The apparent affinity of antagonists did not change when the receptor was radiolabelled with either [3H]-agonists or [3H]-antagonists; however, agonists had a higher apparent affinity for [3H]-agonist-labeled receptors than for [3H]-antagonist-labeled receptors. Therefore, the apparent affinity of agonists for the 5-HT2A receptor estimated from displacement experiments depends on the intrinsic efficacy of the radioligand used.

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.

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.

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.

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.

Neuropeptide Y receptors from calf brain: effect of crude Conus venom preparations on [3H]NPY binding.

NPY receptors are identified in calf frontal cortex and hippocampus membrane preparations by binding of N-[propionyl-3H] neuropeptide Y. Saturation and competition binding data with PYY, NPY-(18-36) and NPY itself fit with a single class of sites: for the radioligand KD = 1.4 +/- 0.5 nM, Bmax = 434 +/- 180 fmol/mg protein in frontal cortex, KD = 0.7 +/- 0.2 nM, Bmax = 267 +/- 50 fmol/mg protein in hippocampus. Competition curves of the Y1-subtype selective agonist [Leu31, Pro34]NPY are biphasic in both membrane preparations: high affinity sites (i.e. Y1-subtype) amount to 80% in frontal cortex and 23% in hippocampus. The remaining sites are of the Y2-subtype. Out of 23 Conus venom preparations, 17 inhibit the binding of [3H]NPY in both membrane preparations, but only two of them (from Conus aulicus and C. pennaceus) do so with high potency (IC50 < 5 micrograms protein/ml). Only one venom preparation (from C. mercator) had weak discriminatory properties (IC50Y2/IC50Y1 = 6). Venom from C. anemone increased the [3H]NPY binding 5-fold and with an IC50 of 15-18 micrograms protein/ml. This binding occurred to the venom itself and was unrelated to the NPY receptors since it was equally potent when displaced by [Leu31, Pro34]NPY, NPY-(18-36), PYY and NPY.

The venom of Conus pennaceus inhibits the binding of [3H]neuropeptide Y by direct interaction with the radioligand.

The venom from the marine snail Conus pennaceus inhibits the binding of [3H]neuropeptide Y to calf brain membranes (Czerwiec et al., 1996a) and, in the present study, also to rat forebrain membranes. These membranes contain about 80% Y1- and 20% Y2-receptors. The inhibition by the venom was concentration-dependent with an IC50 value of 3.4 micrograms ml-1. However, the venom also inhibited the binding of [3H]neuropeptide Y to the glass fibre filters and to the previously discovered ANPY toxin from the venom of Conus anemone (Czerwiec et al., 1996b). This inhibition was related to the ability of one or more of the venom components to bind directly to the radioligand instead of the initially assumed interaction with the neuropeptide Y receptors present in membrane preparations. The complex with Conus pennaceus venom was not retained by the glass fibre filter during the separation of the bound from the unbound [3H]neuropeptide Y. Gel filtration chromatography and denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the active [3H]neuropeptide Y-binding component is likely a approximately 30 kDa polypeptide. Binding of [3H]neuropeptide Y to the venom component(s) was not displaced by 20 microM of the (1-24) N-terminal and the (25-36) C-terminal neuropeptide Y fragments. It is therefore likely that the recognition of the venom component(s) requires both the C- and the N-terminal segments of the neuropeptide Y molecule.

Effect of BIBP3226 on inositol phosphate accumulation and cytosolic calcium level in control and NPY Y1 receptor expressing CHO-K1 cells.

BIBP3226 was developed as a potent, selective and competitive antagonist for NPY Y1 receptors by mimicking the C-terminal part of NPY. In agreement with previous studies, NPY mediated a pertussis toxin sensitive elevation of intracellular calcium concentration in CHO-K1 cells that express recombinant human NPY Y1 receptors which can be inhibited by BIBP3226. Surprisingly micromolar concentrations of BIBP3226 were found to induce by itself a fast increase of intracellular calcium concentration followed by a sustained elevated level of this ion. These responses of BIBP3226 are not mediated by NPY receptor activation since (1) they are still present after NPY receptor activation and desensitization, (2) they are also evoked by the receptor inactive enantiomer BIBP3435, (3) they are not affected by pretreatment of the cells with pertussis toxin, (4) they also occur in non-transfected CHO-K1 cells. Preincubation of the cells with EGTA abolished only the sustained increase calcium concentration elicited by BIBP3226 suggesting that the fast increase of intracellular calcium concentration reflects the mobilization of intracellular calcium pools. The ability of thapsigargin to completely inhibit BIBP3226 mediated responses, in the presence or absence of extracellular calcium indeed indicated that BIBP3226 mobilizes intracellular Ins(1,2,3)P3 sensitive calcium stores. In agreement, BIBP3226 was found to activate phospholipase C since the responses were completely inhibited by U73122. Furthermore, when measured in the presence of 10 mM LiCl, BIBP3226 caused an increased accumulation of inositol phosphates. This effect of BIBP3226 is likely to be mediated by activation of an until now unknown receptor or cellular target that is endogeneously expressed in CHO-K1 cells.

D1 dopamine receptors in human putamen, frontal cortex and calf retina: differences in guanine nucleotide regulation of agonist binding and adenylate cyclase stimulation.

High-affinity binding sites for the D1 dopamine receptor antagonist [3H]SCH 23390 were identified in membranes from human putamen, frontal cortex and calf retina. In frontal cortex binding not only occurred to D1 but also to 5-HT2 receptors. In retina and putamen no binding to 5-HT2 receptors was detected. All further binding experiments in frontal cortex were carried out in the presence of 20 nM mianserin to prevent binding to 5-HT2 receptors. In the 3 tissues, antagonist competition curves were monophasic, whereas competition curves with the agonist dopamine revealed the presence of two binding sites, one having high affinity (RH) (32% in retina, 28% in putamen, and 15% in frontal cortex) and the other having low affinity (RL). In retina, the addition of 100 microM GTP caused a full conversion of RH into RL. In contrast, in frontal cortex, RH sites were not altered by 400 microM GTP or 100 microM 5'-guanylyl-imidodi-phosphate (Gpp(NH)p). In putamen, both guanine nucleotides provoked only a partial conversion of RH into RL. Dopamine (100 microM) produced a 220% and 56% increase in cAMP production in respectively retina and putamen homogenates, while no increase was observed in frontal cortex homogenate. These data may suggest that not all D1-receptors are coupled to the adenylate cyclase system.

Molecular distinction between calf heart and brain muscarine receptors: different N-ethylmaleimide modulation of agonist binding.

Muscarinic acetylcholine receptors in calf heart and forebrain membranes were identified by binding of 1-[benzilic-4,4'-3H]quinuclidinyl benzilate ([3H]QNB). We were able to solubilise these receptors with a yield of 50% of the proteins by treatment of the membranes with digitonin. The existence of two or more receptor subclasses with different agonist affinity in the heart membranes was evidenced by the shallow carbachol/[3H]QNB competition binding curves. The receptors displayed only low agonist affinity, in the presence of GTP as well as after solubilisation. The alkylating reagent N-ethylmaleimide (NEM) caused a 70-fold increase in agonist affinity for the solubilised receptors whereas GTP was ineffective. A similar difference in affinity was observed for the membranes when agonist competition curves in the presence of NEM were compared to those in the presence of GTP. NEM caused only a 2- to 3-fold increase of the agonist affinity for solubilised brain cortex membranes. These data suggest that heart and brain muscarine receptors are structurally different.

High-affinity binding of [3H]neuropeptide Y to a polypeptide from the venom of Conus anemone.

Venom preparation from Conus anemone contains a component that binds radiolabeled neuropeptide Y ([3H]neuropeptide Y) with high affinity (KD = 2.9 nM +/- 0.2 nM, Bmax = 15.2 +/- 0.5 pmol/mg protein). Binding of [3H]neuropeptide Y to the venom component is displaced with nanomolar affinity of unlabeled human and porcine neuropeptide Y, porcine [Leu31-Pro34]neuropeptide Y, peptide YY, avian and bovine pancreatic polypeptide, and the (18-36) and (25-36) C-terminal fragments from neuropeptide Y. No displacement is found with the (1-24) N-terminal neuropeptide Y fragment, human secretin, porcine dynorphin A and Boc-DAKLI (bolton Hunter coupled dynorphin A analog kappa ligand) nor with the non-peptide neuropeptide Y receptor antagonist BIBP3266. Gel filtration chromatography and denaturing (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) show that the [3H]neuropeptide Y-binding component is very likely a single-chain polypeptide with a molecular mass of 18.5 kDa.