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.

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.

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.

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.

High affinity displacement of [(3)H]NPY binding to the crude venom of conus anemone by insect neuropeptides.

The venom from Conus anemone contains a protein, named ANPY toxin, which displayed high affinity (IC(50) in nanomolar range) to neuropeptide Y (NPY), [Leu(31), Pro(34)]NPY, peptide YY, pancreatic polypeptide, the Y(1) antagonist 1229U91, and C-terminal NPY fragments. N-terminal fragments and the free acid form of NPY did not bind to ANPY. The truncated NPY fragments displayed very low affinity to Y(1) receptors and partially inhibited [(3)H]NPY binding to anti-NPY antiserum. Several insect neuropeptides, the sequences of which related to the C-terminal fragments of NPY, were observed to bind with similar affinity or even 20 times higher (Lom-MS and Scg-NPF) affinity than NPY. In contrast, no significant binding of these insect peptides was observed for Y(1) receptors and anti-NPY antiserum. Therefore, ANPY can be viewed as an acceptor that binds with very high affinity to a broad spectrum of vertebrate and invertebrate neuropeptides that share a similar C-terminal amino acid sequence.

Identification of I1 and I2 imidazoline receptors in striatum membranes from different species.

The alpha 2-adrenergic agonist [3H]clonidine and antagonist [3H]idazoxan also label I1 and I2 imidazoline receptors in striatum membranes. They are investigated here in striata from the dog, rat, mouse, rabbit, calf, monkey, and human. I1 receptors were barely detected in the dog, rat, and mouse and only further examined by competition binding experiments in calf, rabbit, and human. I2 receptors were further examined in all species. The centrally acting vasodilators clonidine and rilmenidine were more potent than moxonidine at the I1 receptors. They displayed low potency for the I2 receptors in all species except the rat. In all species examined, the nonsubstituted imidazoline derivatives idazoxan and RX801077 displayed high affinity for the I1 and I2 receptors. Conversely, both stereoisomers of the alkoxy-substituted imidazoline-derivative efaroxan displayed low affinity. The matching binding characteristics of these compounds further stress the structural similarity of the ligand binding sites of I1 and I2 receptors.

Insurmountable angiotensin AT1 receptor antagonists: the role of tight antagonist binding.

Angiotensin II increased the inositol phosphates production (EC50 = 3.4+/-0.7 nM) in Chinese hamster ovary (CHO) cells expressing the cloned human angiotensin AT1 receptor (CHO-AT1 cells). Coincubation with angiotensin AT1 receptor antagonists produced parallel rightward shifts of the concentration-response curve without affecting the maximal response. The potency order is 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benz imidazoline-7-carboxylic acid (candesartan) > 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]i midazole-5-carboxylic acid (EXP3174) > 2-n-butyl-4-spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)methyl]2-imidazolin-5-one (irbesartan)> of 2-n-butyl-4-chloro-5-hydroxymethyl-1-(2'-(1H-tetrazol-5-yl)bipheny l-4-yl)methyl]imidazole (losartan). Additionally, preincubation with these antagonists depressed the maximal response, i.e., 95%, 70%, 30% of the control response for candesartan, EXP3174 and irbesartan and not detectable for losartan. Increasing the antagonist concentration or prolonging the preincubation time did not affect this depression. Furthermore, these values remained constant for candesartan and EXP3174, when the angiotensin II incubation time varied between 1 and 5 min. Our data indicate that antagonist-receptor complexes are divided into a fast reversible/surmountable population and a tight binding/insurmountable population at the very onset of the incubation with angiotensin II.

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.

Binding of the antagonist [3H]candesartan to angiotensin II AT1 receptor-transfected [correction of tranfected] Chinese hamster ovary cells.

Binding of the non-peptide angiotensin II AT1 antagonist [3H](2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]- H-benzimidazoline-7-carboxylic acid ([3H]candesartan) to human angiotensin II AT1 receptor-transfected Chinese hamster ovary (CHO-AT1) cells was inhibited to the same extent by angiotensin II and non-peptide angiotensin II AT1 antagonists. No binding was observed in control CHO-K1 cells. Dissociation was slow (k(-1) = 0.0010+/-0.0001 min(-1)) after removal of the free [3H]candesartan but increased 5-fold upon addition of supramaximal concentrations of angiotensin II AT1 antagonists. Angiotensin II responses recovered equally slow from candesartan-pretreatment. When washed and further incubated, these angiotensin II responses also recovered more rapidly in the presence of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphen yl-4-yl)methyl]imidazole (losartan), indicating that unlabelled ligands prevented reassociation. [3 H]candesartan saturation binding experiments required a long time to reach equilibrium. Therefore, the equilibrium dissociation constant (Kd = 51+/-8 pM) was calculated from the association and dissociation rate constants. Our findings indicate that the insurmountable nature of candesartan in functional studies is related to its slow dissociation from the receptor.

The effects of candesartan on human AT1 receptor-expressing Chinese hamster ovary cells.

Chinese hamster ovary cells expressing the cloned human angiotensin II receptor of the AT1 subtype (CHO-AT1 cells) were used as an 'in vitro" model system to investigate the action mechanism of the nonpeptide AT1 receptor blocker candesartan. In the presence of 10 mM LiCl, angiotensin II causes a long-lasting increase in the production of inositol phosphates in these cells. This effect is dose-dependent with half-maximal stimulation (EC50) at 3 nM angiotensin II. Pre-incubation of the cells for 30 min at 37 degrees C with candesartan decreases the maximal response to angiotensin II by up to 90%, with a half-maximal decrease at 0.5 nM candesartan. At this concentration, candesartan only produces a slight rightward shift of the angiotensin II dose-response curve. Recovery experiments on CHO-AT1 cells reveal that the inhibitory effect of candesartan is only slowly reversed after removal of the blocker. The insurmountable effect of candesartan can therefore be ascribed to its long-lasting inhibition of the AT1 receptor.

Human neuropeptide YY1 receptors exert unequal control of the extracellular acidification rate in different cell lines.

The ability of the human neuropeptide YY1 receptor subtype to increase the extracellular acidification rate in different cell lines was investigated by using the Cytosensor Microphysiometer. In CHO-Y1 cells (Chinese Hamster Ovary cells expressing the cloned human neuropeptide YY1 receptor), neuropeptide Y increased the acidification rate by up to 15% of the basal level with a -Log(EC50) of 7.42. As expected for neuropeptide YY1 receptors, this response was potently inhibited by the neuropeptide YY1-selective non-peptide antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginine amide). Its enantiomer BIBP3435 ((S)-N2-(diphenylacetyl)-N-[(4-hydroxy-phenyl)methyl]-D-arginin amide) was less potent. The antagonists themselves did not affect the extracellular acidification rate at concentrations up to 10 microM. In SK-N-MC cells (a neuroblastoma cell line of human origin that expresses the neuropeptide YY1 receptor) no change of the acidification rate could be observed in the presence of neuropeptide Y at concentrations up to 1 microM. For control, the neuropeptide YY1 receptors were also investigated by assessing whole cell radioligand binding and, at the functional level, by assessing their ability to decrease the forskolin-induced accumulation of cAMP. The specific (i.e., neuropeptide Y-displaceable) binding of [3H]neuropeptide Y was to a homogeneous class of high-affinity sites in both SK-N-MC and CHO-Y1 cells. The equilibrium dissociation constants for [3H]neuropeptide Y, the total number of binding sites and the kinetic constants for association and for dissociation were similar. Neuropeptide Y produced a dose-dependent inhibition of forskolin-induced cAMP accumulation in SK-N-MC cells (-log(EC50) = 9.40) but it did not affect cAMP accumulation in CHO-Y1 cells. Non-transfected CHO-K1 cells were used as negative control throughout the study. No binding or response could be observed in these cells. Our data suggest that the signalling mechanisms of neuropeptide YY1 receptors are closely related to the cell type in which they are expressed.

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.

[3H]-BIBP3226 and [3H]-NPY binding to intact SK-N-MC cells and CHO cells expressing the human Y1 receptor.

We have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80% of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged.

Isoquinoline derivatives isolated from the fruit of Annona muricata as 5-HTergic 5-HT1A receptor agonists in rats: unexploited antidepressive (lead) products.

The fruit and the leaves of Annona muricata (Annonaceae) are used in traditional medicine for their tranquillizing and sedative properties. Extracts of the plant have been shown to inhibit binding of [3H]rauwolscine to 5-HTergic 5-HT1A receptors in calf hippocampus, and three alkaloids, annonaine (1), nornuciferine (2) and asimilobine (3), isolated from the fruit have been shown to have IC50 values of 3 microM, 9 microM and 5 microM, respectively, although in ligand-binding studies it was not possible to determine whether interaction of these ligands with the receptor was agonistic or antagonistic. This paper presents the results of functional assays of the alkaloids. The inhibition of cAMP accumulation was tested in NIH-3T3 cells stably transfected with the 5-HT1A receptor from man. None of the alkaloids showed antagonistic properties towards the 5-HT1A receptors because in the antagonistic tests no influence on the forskolin-stimulated increase of cAMP level was detected. Full agonistic properties were measured for all three compounds; the inhibition constants (Ki) for 1, 2 and 3 were < 10 microM. Inhibition of the binding of the radioligand to the 5-HT1A receptor was observed in every ligand-binding assay performed with the alkaloids; the Ki values for 1, 2 and 3 were in the microM range. These results imply that the fruit of Annona muricata possesses anti-depressive effects, possibly induced by compounds 1, 2 and 3, and that in the past potent leads for the development of anti-depressive therapeutics have not been used.

Cirsimarin and cirsimaritin, flavonoids of Microtea debilis (Phytolaccaceae) with adenosine antagonistic properties in rats: leads for new therapeutics in acute renal failure.

In traditional medicine Microtea debilis is used against proteinuria. In ligand-binding studies extracts of Microtea debilis have been shown to inhibit the binding of [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) to adenosine-A1 receptors in rat forebrain membranes. Subsequently, cirsimarin, a flavonoid, was isolated as the active component and was shown to function as adenosine antagonist at the adenosine-A1 receptor in-vitro. In this study we have investigated the adenosine-A2 receptor activity of cirsimarin the in-vivo inhibition of the effects of adenosine by cirsimarin in rats, the absorption of cirsimarin and the inhibition of the binding of [3H]DPCPX to the adenosine-A1 receptor by urine samples obtained after oral administration of crude extract of Microtea debilis, cirsimarin or cirsimaritin to rats. Cirsimarin inhibited the binding of [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) to adenosine-A2 receptors in rat striatum with an inhibition constant, Ki, of 6.5 +/- 0.3 microM. The decrease of heart rate and blood pressure induced by adenosine was significantly inhibited by cirsimarin. After oral administration of 8 and 80 mg kg-1 cirsimarin, the compound could not be detected in either plasma or urine, but the presence of cirsimaritin was established. By use of beta-glucuronidase, glucuronides of cirsimaritin were also detected in the urine. The concentrations of cirsimaritin in the plasma were 0.126 +/- 0.04, 0.138 +/- 0.015, and 0.120 +/- 0.022 microM, respectively, 2, 5 and 12 h after administration of 8 mg kg-1 cirsimarin. The concentrations of cirsimaritin in the urine at the same times after administration of the same dose were 2.05 +/- 1.86, 5.05 +/- 2.6 and 2.06 +/- 0.09 microM, respectively. The inhibition of the binding of [3H]DPCPX to the adenosine-A1 receptor by urine samples collected 2, 5 and 12 h after oral administration of 8 mg kg-1 cirsimarin or a crude extract of Microtea debilis containing approximately 8 mg kg-1 cirsimarin and 2.8 mg kg-1 cirsimaritin, or 6.8 mg kg-1 cirsimaritin, was not significantly different from that of urine samples collected from untreated rats, in contrast with urine samples collected 1 and 2 days after oral administration of 80 mg kg-1 cirsimarin. Approximately 3% of the cirsimarin was excreted in the urine as cirsimaritin. The results indicate that in the kidney and urinary tract the concentrations of cirsimaritin produced after ingestion of more than 8 mg kg-1 cirsimarin can be high enough to inhibit the interaction of adenosine with its receptors; this might explain the effectiveness of Microtea debilis preparations against proteinuria in traditional medicine.

The novel alpha 2-adrenoceptor agonist [3H]mivazerol binds to non-adrenergic binding sites in human striatum membranes that are distinct from imidazoline receptors.

The alpha 2 adrenergic agonist [3H]mivazerol labelled two populations of binding sites in membranes from the human striatum. Forty per cent of the sites labelled by 3 nM [3H]mivazerol corresponded to alpha 2 adrenergic receptors as they displayed a high affinity for (-)-adrenaline and for rauwolscine. The remaining binding was displaced by mivazerol with a pIC50 of 6.5 +/- 0.1. These sites displayed higher affinity for dexmedetomidine (pIC50 = 7.1 +/- 0.1), but much lower affinity for clonidine (pIC50 < 5.0) and for idazoxan (pIC50 = 5.1 +/- 0.1). Mivazerol also showed low affinity for the [3H]clonidine-labelled I1 imidazoline receptors and for the [3H]idazoxan-labelled I2 receptors (pIC50 = 5.1 and 3.9, respectively). These results suggest that the non-adrenergic [3H]mivazerol binding sites are distinct from the imidazoline receptors in the human striatum.

Non-competitive binding of the nonpeptide antagonist BIBP3226 to rat forebrain neuropeptide Y1 receptors.

[3H]Neuropeptide Y labelled neuropeptide Y receptors in rat forebrain membranes as a homogenous class of high-affinity sites. Between 80 and 85% of these receptors showed high affinity for Y1-selective antagonists such as (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine amide (BIBP3226). While competitive in functional studies, BIBP3226 produced parallel shifts of the Scatchard plots of [3H]neuropeptide Y saturation binding in rat forebrain membranes. Mechanisms which are routinely invoked to explain non-competitive binding do not apply to BIBP3226. Wash-out experiments, involving successive treatment of the membranes with BIBP3226, buffer (wash-out step) and [3H]neuropeptide Y, argue against irreversible or a pseudo-irreversible binding of the antagonist. Allosteric inhibition is also unlikely since BIBP3226 did not affect the rate of dissociation of [3H]neuropeptide Y in isotope dilution experiments. The non-hydrolyzable guanine nucleotide, 5'-guanylylimidodiphosphate (Gpp(NH)p), abolished the binding of [3H]neuropeptide Y and increased its rate of dissociation in isotope dilution experiments. This suggests that the initial [3H]neuropeptide Y-receptor association is a low affinity process and that the observed binding of [3H]neuropeptide Y is related to the formation of a ternary [3H]neuropeptide Y-receptor-G protein complex. Two- or even multistate models (in which BIBP3226 could potentially behave as an inverse agonist) could therefore be needed to explain the non-competitive antagonism of BIBP3226 in broken cell preparations.

Adenosine-1 active ligands: cirsimarin, a flavone glycoside from Microtea debilis.

Several plants collected through different approaches were screened on distinct receptors using ligand-binding studies as bioassay. Extracts of Microtea debilis showed high activity on adenosine A1 receptors. Bioassay-guided fractionation using ligand-binding studies resulted in the isolation of an adenosine A1 active ligand, cirsimarin (cirsimaritin 4'-O-glucoside). GTP did not influence the radioligand inhibition curve of cirsimarin, indicating that this compound is acting as an antagonist at the adenosine-A1 receptors. The use of this plant against "proteinuria" in traditional medicine in Suriname (South America) may be explained by the adenosine A1 antagonistic action of cirsimarin. A series of flavonoids was tested in the same assay, but they were less active. No structure-activity relationship could be observed.

Pharmacological characterization of I1 and I2 imidazoline receptors in human striatum.

[3H]RX821002, [3H]clonidine and [3H]idazoxan have previously been shown to selectively label alpha 2-adrenergic receptors, I1 and I2 imidazoline receptors in the human central nervous system, respectively. Idazoxan shows relatively high affinity for all three receptors. We investigated the possible selectivity of several compounds towards one of those receptors in human striatum. Addition of an alkoxy group at the 2-position of the benzodioxan moiety of idazoxan (ethoxy-idazoxan, methoxy-idazoxan) increases the alpha 2-selectivity in human brain. Efaroxan is also alpha 2-selective. On the contrary, BU224, BU239, cirazoline and RX801077 display imidazoline receptor selectivity. Our results indicate that for all molecules tested, idazoxan and 'flat' analogs possess I1/I2 receptor selectivity. A 'bulky' substituent at the 2-position of the benzodioxan ring gives rise to alpha 2-adrenergic receptor selectivity. Until now, we found no more than 3-fold difference in IC50 between both imidazoline receptors. Both receptors also display similar stereoselectivity, suggesting that they might be 'interconnected' in the human striatum.

Medicinal plants in Suriname: screening of plant extracts for receptorbinding activity.

Ligand-binding-studies on twelve different receptors were used for screening extracts from plants from Suriname (South-America). The results on 5-HT(1A), A(1) and NMDA receptors promote further investigation of Microtea debilis, Hibiscus bifurcatus, Irlbarchia purpurascens, Miconia ciliata and Mimosa myriadena.