Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 μM) results in the inhibition of Ca²⁺-dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 μM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [³H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.     

Atypical nuclear localization of VIP receptors in glioma cell lines and patients

An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, by applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.     

The postnatal development of VIP binding sites in rat forebrain and hindbrain

The specific binding of 125I-labeled vasoactive intestinal peptide (VIP) to brain membranes from the forebrain and hindbrain regions of 2 to 37-day postnatal rats was measured. In both regions of the brain, VIP binding was low but detectable two days after birth and rose markedly between postnatal days 7 and 17. This increase in VIP binding with age correlates well with observed increases in VIP-stimulated adenylate cyclase activity and increases in VIP content as determined by radioimmunoassay. In hindbrain, the density of VIP binding sites was substantially higher than the forebrain at two days, while at 37 days, they were about equal, suggesting that the hindbrain regions may mature neurochemically prior to the forebrain. Total binding sites for forebrain and hindbrain were about equal at birth for both brain regions, while forebrain had a substantially greater number of sites at 37 days postnatal. The presence of VIP binding sites in both forebrain and hindbrain early in postnatal development suggests that VIP may play a role in development of the brain.     

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.     

Development of dry powder inhalation system of novel vasoactive intestinal peptide (VIP) analogue for pulmonary administration

Vasoactive intestinal peptide (VIP) exerts a relaxing action on tracheal smooth muscle which is mediated through interaction with VIP receptors. The deficiency of VIP in the airways has been implicated in the pathogenesis of asthma. Thus, the administration of VIP may be useful for the therapy of pulmonary diseases. However, the therapeutic application of VIP is largely limited by its rapid degradation in addition to the systemic adverse effects due to the wide distribution of VIP receptors. To overcome these problems, we succeeded to synthesize a novel VIP derivative of VIP, [R15, 20, 21, L17]-VIP-GRR (IK312532), and to prepare its dry powder for the topical administration to the lung. The physicochemical properties of dry powder were evaluated by laser diffraction and cascade impactor. The laser diffraction analysis indicated that the carrier and fine particles had median diameter of 65.6 and 4.5 microm, respectively, and the air flow at the pressure of 0.15 MPa or higher resulted in the high dispersion and significant separation of fine particle containing peptide from the carrier molecule. The cascade impactor analysis clearly showed the high emission of dry powder from capsule and the deposition of peptide on stages 3 of the cascade impactor. The intratracheal administration of dry powder inhaler (DPI) of VIP or IK312532 brought about a significant decrease of maximal number of binding sites (Bmax) for [125I]VIP in anterior and posterior lobes of rat right lung, suggesting a significant occupancy of lung VIP receptors. This effect by IK312532-DPI compared with VIP-DPI lasted for a longer period. Thus, IK312532-DPI may be a pharmacologically useful drug delivery system for the VIP therapy of pulmonary diseases such as asthma.     

Characterization of insect neuronal octopamine receptors (OA3 receptors)

Octopamine receptors in the nervous tissue of insects were investigated using a ligand-receptor assay with [³H]NC-5Z or [³H]octopamine as the radioligands. Both ligands recognized a homogenous class of binding sites with the properties of an octopamine receptor. This receptor has been characterized pharmacologically. Both high-affinity agonists (e.g. NC 7, K₁=0.3 nM) and antagonists (e.g. maroxepine, K₁=1.02 nM) were investigated. The neuronal octopamine receptor belongs to a receptor class that can easily be distinguished from peripheral octopamine receptors. Initial investigations of the localization of octopamine receptors within the insect nervous tissue show the greatest receptor density in the optic lobes.     

The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca⁺-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca²⁺ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.     

G protein-coupled receptors in energy homeostasis

G-protein coupled receptors (GPCRs) compromise the largest membrane protein superfamily which play vital roles in physiological and pathophysiological processes including energy homeostasis. Moreover, they also represent the up-to-date most successful drug target. The gut hormone GPCRs, such as glucagon receptor and GLP-1 receptor, have been intensively studied for their roles in metabolism and respective drugs have developed for the treatment of metabolic diseases such as type 2 diabetes (T2D). Along with the advances of biomedical research, more GPCRs have been found to play important roles in the regulation of energy homeostasis from nutrient sensing, appetite control to glucose and fatty acid metabolism with various mechanisms. The investigation of their biological functions will not only improve our understanding of how our body keeps the balance of energy intake and expenditure, but also highlight the possible drug targets for the treatment of metabolic diseases. The present review summarizes GPCRs involved in the energy control with special emphasis on their pathophysiological roles in metabolic diseases and hopefully triggers more intensive and systematic investigations in the field so that a comprehensive network control of energy homeostasis will be revealed, and better drugs will be developed in the foreseeable future.     

Steroids and receptors in canine mammary cancer

The aims of this study were to investigate the serum and tissue content of androgens and estrogens in canine inflammatory mammary carcinomas (IMC) as well as in non-inflammatory malignant mammary tumors (MMT), and assessed the immunoexpression of estrogen and androgen receptors using immunohistochemistry. Profiles for the androgens dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T), and for the estrogens 17beta estradiol (E2) and estrone-sulphate (SO4E1) were measured both in tissue homogenates and in serum of MMT and IMC by EIA techniques in 42 non-inflammatory malignant mammary tumors (MMT) and in 14 inflammatory mammary carcinomas (IMC), prospectively collected from 56 female dogs. Androgen receptor (AR) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression was studied using immunohistochemistry (strepavidin-biotin-peroxidase method) in samples of 32 MMT and 14 IMC, and counted by a computer image analyzer. IMC serum and tissue levels of androgens were significantly higher than MMT levels. Tissue content of estrogens was also significantly higher in IMC than in MMT. Serum values of SO4E1 were significantly higher in IMC, but serum levels of E2 were significantly lower in IMC compared to MMT cases. Medium-high androgen receptor intensity was observed in 64.28% of IMC and 40.62% of MMT. No important differences were found between ERalpha expression in IMC (100% negative) and MMT (90% negative). ERbeta and AR were intensely expressed in highly malignant inflammatory mammary carcinoma cells. To our knowledge, this is the first report relative to AR immunohistochemistry in canine mammary cancer and to estrogens or androgens in serum of dogs with benign or malignant mammary tumors.     

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line

R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [¹²⁵I]-PACAP or [¹²⁵I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC₅₀ of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC₅₀ in agreement with the pharmacological profile of the VPAC₁ receptor subtype: PACAP = VIP > [K¹⁵, R^16, L²⁷]VIP(1–7)/GRF(8–27) = [R¹⁶]ChSn (two VPAC₁ agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC₂ receptor was inactive at 1 μM. Dose-response curves of VPAC₁ agonist molecules (PACAP, VIP, [K¹⁵, R¹⁶, L²⁷]VIP(1–7)/GRF(8–27), [R¹⁶]ChSn) were shifted to the right by the VPAC₁ receptor antagonist [AcHis¹, D-Phe², Lys¹⁵, Leu¹⁷]VIP(3–7)/GRF(8–27), with a K_i of 3 ± 1 nM (n = 3). The presence of VPAC₁ receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC₁ receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC₁ receptors undergoing rapid desensitization in HEL cells.     

Comparative efficacy of VIP and analogs on activation and internalization of the recombinant VPAC2 receptor expressed in CHO cells

Using a monoclonal antibody interacting with the extracellular amino-terminus of the human VPAC₂ receptor but that did not interfere with ligand binding, we measured by flow cytometry receptor internalization and trafficking induced by full agonists, partial agonists and an antagonist in Chinese hamster ovary cells expressing the recombinant receptor. The agonists, but not the antagonist, induced a rapid, dose-dependent receptor internalization blocked by hypertonic sucrose that was more pronounced for the VIP analog N-hexanoyl-VIP (80%) than for VIP and Ro 25-1553 (50%) and the [A¹¹]-VIP (20%). Re-expression of the receptors at the membrane was achieved within two hours after exposure to VIP and Ro 25-1553 was blocked by 25 μM monensin but not by 10 μg/ml cycloheximide. Re-expression was much slower after exposure to the acylated peptide and was blocked by preincubation with 25 μM monensin and 10 μg/ml cycloheximide.     

孤儿G蛋白偶联受体研究进展

孤儿G蛋白偶联受体的研究意味着发现其尚未了解的内源性配体,是后基因组时代功能基因组学研究的热点之一,对生命科学的发展具有深 影响。本文介绍孤儿G蛋白偶联受体的概念、研究策略及其应用。     

Characterization of VIP- and helodermin-preferring receptors on human small cell lung carcinoma cell lines

We investigated the molecular and pharmacologic characteristics of VIP receptors on two human SCLC cell lines: NCI-N592 and NCI-H345. With NCI-N592 cell, the order of potency of VIP-related peptides in inhibiting ¹²⁵I-VIP binding and in stimulating cAMP production was typical of the human VIP receptor. By covalent cross-linking, a polypeptide of Mr 62,300 was obtained. Conversely, the behavior of NCI-H345 cell line was totally different: helodermin was the most potent peptide, VIP and PHI were equipotent, while hGRF and secretin were totally ineffective. These results suggest that NCI-N592 cells possess a typical VIP receptor while NCI-H345 cells possess a helodermin-preferring receptor, and that the natural target of helodermin might not be the VIP receptor.     

The effects of experimental uremia in rats on duodenal VIP levels and the interaction of VIP with duodenal epithelial cells

The effects of experimental uremia on the concentration of vasoactive intestinal peptide (VIP) in duodenum as well as on the interaction of this neuropeptide with the corresponding epithelial cells were studied in rats. Duodenal VIP concentration was significantly decreased in uremic rats as compared to control animals. The specific binding of VIP to duodenal epithelial cells increased in rats with uremia due to an increase in the number of VIP receptors rather than a change in the binding affinity or in the extent of VIP degradation. On the other hand, the efficacy but not the potency of VIP upon cyclic AMP generation varied in parallel to that observed at the receptor level.     

Structural characterization of intracellular C-terminal domains of group III metabotropic glutamate receptors

Metabotropic glutamate receptors (mGluRs) are regulated by interacting proteins that mostly bind to their intracellular C-termini. Here, we investigated if mGluR6, mGluR7a and mGluR8a C-termini form predefined binding surfaces or if they were rather unstructured. Limited tryptic digest of purified peptides argued against the formation of stable globular folds. Circular dichroism, ¹H NMR and ¹H¹⁵N HSQC spectra indicated the absence of rigid secondary structure elements. Furthermore, we localized short linear binding motifs in the unstructured receptor domains. Our data provide evidence that protein interactions of the analyzed mGluR C-termini are mediated rather by short linear motifs than by preformed folds.     

The effect of thiol reagents on GABA transport in rat brain synaptosomes

The nature of gamma-aminobutyric acid (GABA) transport has been investigated in preparations of rat brain synaptosomes using a number of thiol reagents with varying membrane permeabilities. N-Ethylmaleimide, p-chloromercuribenzoate and p-chloromercuriphenylsulfonate effectively inhibited GABA transport in both directions (i.e., uptake and release) whereas 5,5'-dithiobis-2-nitrobenzoate, mercaptopropionate and N- nitroethylenediamine were much less effective, or ineffective, even at millimolar concentrations. For each of the thiol reagents, the inhibition profile for GABA uptake was approximately the same as that for its release. The effectiveness of the reagents indicates that there is an external, reactable SH-group on the transporter, that the thiol reagent must be somewhat lipophilic for it to react with the SH-group(s), and that the same synaptosomal transport system is responsible for both uptake and release of GABA.     

Modelling the activation of G-protein coupled receptors by a single drug

In this paper, the most popular proposed mechanism for activation of G-protein coupled receptors (GPCRs) - the shuttling mechanism - is modelled mathematically. An asymptotic analysis of this model clarifies the dynamics of the system in the presence of a drug, in particular identifying which reactions dominate during the different timescales. The modelling also reveals challenging behaviour in the form of a peak response. This new mechanism gives simple explanations for complex, possibly misunderstood, behaviour.     

VIP receptor antagonists inhibit mammary carcinogenesis in C3(1)SV40T antigen mice

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist on mammary carcinogenesis were investigated using the C3(1)SV40T antigen (ag) mice. Ten microg/day VIPhybrid (VIPhyb) administered daily subcutaneously increased significantly the survival of C3(1)SV40Tag mice. At 5.2 months, VIPhyb significantly reduced the mammary tumor burden in C3(1)SV40Tag mice relative to control animals. 125I-VIP bound with high affinity to mouse mammary tumor homogenate. Because (Lys15, Arg16, Leu27)VIP1-7GRF8-27 (VPAC1 selective) but not Ro25-1553 (VPAC2 selective) inhibited specific 125I-VIP binding to mammary tumor membranes with high affinity, VPAC1 receptors predominate. By RT-PCR, VPAC1 receptor mRNA was detected in mammary tumors. By Western blot, a major 60 Kdalton band was detected in mammary tumor extracts using VPAC1 receptor antisera. By immunocytochemistry, VPAC1-R immunostaining was detected in the cytosol and plasma membrane but not the nucleus of fixed mammary tumor tissue. Using laser capture microdissected tumor cells and surface enhanced laser desorption/ionization (SELDI) techniques on mammary tumor cells, the proteomic profile was altered in mice treated with VIPhyb. Because VPAC1 receptor antagonists increase the survival and reduce the tumor burden in C3(1)SV40Tag mice, they may function as chemopreventive agents in mammary cancer.     

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 μM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. B_max of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 μM VIP. No significant change was observed in receptor affinity, in B_max of the low-affinity receptor, in ED₅₀, or in ED₁₀₀ of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 μM, 3 h) caused 24% reduction in [¹²⁵I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 μM, 3 h).