Conformational analysis of pseudo-peptides: The case of FK888, a potent and selective substance P receptor antagonist

Summary Molecular recognition is a central problem in medicinal sciences, and therefore a knowledge of the salient molecular features neccessary for efficient interaction with a receptor as well as their relative spatial arrangement is of crucial importance. Thus, an insight into probable biorelevant 3D structures by conformational analysis is equally fundamental. In the present study, we describe the conformational analysis of FK888, a potent and selective pseudo-peptide antagonist of the NK₁ receptor of substance P, using an in-house developed method (CONFEX: CONFormational EXploration). Conformations could be subdivided into four families according to peptidic folding: the first two present an extended conformation which can be characterized as a hairpin-like structure, while the other two present a β-turn-like arrangement. These results were compared with experimental findings obtained by NMR spectroscopy.     

Comparative conformational analysis of two endothelin-B antagonists

Summary A comparative conformational analysis of two short pseudopeptides with ET_B receptor affinity has been performed by molecular modelling and NMR techniques. This analysis aimed to get insight into probable biorelevant conformations and pharmacophoric patterns necessary for an efficient interaction with the receptor. Thus, it was shown that the two compounds can adopt -turn (or -turn-like) conformations, based on which the synthesis of particular, more rigid analogs might be proposed. The results obtained should prove valuable in a strategy aiming to design new endothelin antagonists following a peptide to non-peptide approach.     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

Genetic analysis using trans-dominant linked markers in an F2 family

Trans-dominant linked markers pairs (trans referring to the repulsion linkage phase) provide a model for inferring the F₂ progeny genotype based upon both the conditional probabilities of F₂ genotypes, given the F₂ phenotype, and prior information on marker arrangement. Prior information of marker arrangement can be readily obtained from a linkage analysis performed on marker segregation data in a family resulting by crossing the F₁ individual to a tester parent or else can be obtained directly from the gametes of the F₁, or from recombinant inbred lines. We showed that a trans-dominant linked marker (TDLM) pair can be recoded as a co-dominant megalocus when the recombination fraction, r₁, for apair of TDLMs is less than 0.05. We obtained a maximum-likelihood estimator (MLE) of the recombination frequency, r₂, between a TDLM pair and a co-dominant marker in an F₂ family using the EM algorithm. The MLE was biased. Mean bias increased as r₁ and r₂ increased, and decreased as sample size increased. The information content for r₂ was compared to the information content of dominant and co-dominant markers segregating in an F₂ family. It was almost identical with two co-dominant markers when r₁0.01 and r₂0.05. For larger values of r₁, (0.05r₁0.15) a TDLM pair provided 75%–66% of the information content of two co-dominant markers. Although dominant markers can be converted to co-dominant markers by a laborious process of cloning, sequencing, and PCR, TDLM pairs could easily substitute for co-dominant markers in order to detect quantitative trait loci (QTLs) and estimate gene action in an F₂ family.     

Hydroxy-1,2,5-oxadiazolyl moiety as bioisoster of the carboxy function. A computational study on γ-aminobutyric acid (GABA) related compounds

Recently, our research group has proposed the hydroxyfurazanyl (4-hydroxy-1,2,5-oxadiazole-3-yl) moiety as a new non-classical isoster of the carboxy function in the design of γ-aminobutyric acid (GABA) analogues. Some compounds showed significant activity at the GABA_A receptor, representing the only examples of pentatomic heterocycles bearing an ω-aminoalkyl flexible side chain in the position vicinal to the hydroxy group displaying agonist activity at this receptor subtype. In this work, an ab initio analysis of the structural and electronic features of furazan-3-ol is presented, in order to provide a theoretical basis to the claimed bioisosterism with the carboxy function. An ab initio conformational study with the C-PCM implicit solvent model was carried out to elucidate the reasons of the peculiar behaviour of the furazan models. Alongside, another conformational search through molecular dynamics in explicit solvent was accomplished, in order to validate the first method. The electronic features of the 4-hydroxy-1,2,5-oxadiazole-3-yl substructure seem to account for a marked stabilising effect of the putative bioactive conformation at the GABA_A receptor subtype. The 1,2,5-thiadiazole analogue, which shares the same conformational preference of its oxygenated counterpart, was identified as a potential candidate for synthesis and pharmacological testing. Figure 4-(ω-aminoalkyl)-1,2,5-oxadiazole-3-ol analogues of GABA Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Nuclease S1 analysis of eubacterial 5S rRNA secondary structure

Summary Single-strand-specific nuclease S₁ was employed as a structural probe to confirm locations of unpaired nucleotide bases in 5S rRNAs purified from prokaryotic species of rRNA superfamily I. Limited nuclease S₁ digests of 3- and 5-end-labeled [³²P]5S rRNAs were electrophoresed in parallel with reference endoribonuclease digests on thin allel with reference endoribonuclease digests on thin sequencing gels. Nuclease S₁ primary hydrolysis patterns were comparable for 5S rRNAs prepared from all 11 species examined in this study. The locations of base-paired regions determined by enzymatic analysis corroborate the general features of the proposed universal five-helix model for prokaryotic 5S rRNA, although the results of this study suggest a significant difference between prokaryotic and eukaryotic 5S rRNAs in the evolution of helix IV. Furthermore, the extent of base-pairing predicted by helix IV needs to be reevaluated for eubacterial species. Clipping patterns in helices II and IV appear to be consistent with a secondary structural model that undergoes a conformational rearrangement between two (or more) structures. Primary clipping patterns in the helix II region, obtained by S₁ analysis, may provide useful information concerning the tertiary structure of the 5S rRNA molecule.     

The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

Background

Dopamine signaling is mediated by G_s protein-coupled “D₁-like” receptors (D₁ and D₅) and G_i-coupled “D₂-like” receptors (D_2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the G_i-coupled dopamine D₂ receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the G_s-coupled dopamine D₁-like receptor subtypes have never been identified on these cells. Activation of G_s-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D₁-like receptor is expressed on ASM, and modulates its function through G_s-coupling.

Methods

The mRNA and protein expression of dopamine D₁-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D₁ receptor, HASM cells were treated with dopamine or the dopamine D₁-like receptor agonists ({"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 or {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D₁ receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BK_Ca) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576.

Results

Messenger RNA encoding the dopamine D₁ and D₅ receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D₁ receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D₁ receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D₁-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D₁-like receptor antagonists {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 or {"type":"entrez-protein","attrs":{"text":"SCH39166","term_id":"1052842517","term_text":"SCH39166"}}SCH39166. {"type":"entrez-nucleotide","attrs":{"text":"A68930","term_id":"4759850","term_text":"A68930"}}A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576.

Conclusions

These results demonstrate that the dopamine D₁ receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.     

A new D₂ dopamine receptor agonist allosterically modulates A(2A) adenosine receptor signalling by interacting with the A(2A)/D₂ receptor heteromer

The structural and functional interaction between D₂ dopamine receptor (DR) and A_2A adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinson's disease. In transfected cell lines it has been demonstrated the activation of D₂DR induces a significant negative regulation of A_2AAR-mediated responses, whereas few data are at now available about the regulation of A_2AAR by D₂DR agonists at receptor recognition site. In this work we confirmed that in A_2AAR/D₂DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D₂DR agonists were able to negatively modulate both A_2AAR affinity and functionality. These effects occurred even if any significant changes in A_2AAR/D₂DR energy transfer interaction could be detected in BRET experiments.Since the development of new molecules able to target A_2A/D₂ dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D₂DR and modulator of A_2A-D₂ receptor dimer. This compound was able to negatively modulate A_2AAR binding properties and functional responsiveness in a manner comparable to classical D₂R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D₂DR homomers and heteromers and induced A_2AAR/D₂DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A_2AAR/D₂ DR heteromers.     

Effect of light and gibberellic acid on cell division in the first foliage leaf of durum wheat (Triticum durum Desf.)

The present paper is part of a research program which aims at a quantitative analysis of the effects of light and gibberellic acid (GA₃) on growth of the first foliage leaf in durum wheat (Triticum durum Desf.). Since leaf growth is the combined result of the increase in cell number (cell division) and cell enlargement, the influence of light and GA₃ treatment on cell division in the basal meristem of the first leaf in two cultivars, Cappelli and Creso, was investigated. Creso is a short-strawed cultivar carrying the Gai 1 gene which influences both plant height and insensitivity to applied GA₃. Cell division, as measured by mitotic index, was similar in darkness, continuous red light and dichromatic irradiation (far-red plus red), while lower mitotic rates were observed under continuous far-red light: this indicates that the response of cell division is modulated by a high-irradiance reaction of phytochrome in both cultivars. The two cultivars showed different responses to blue light. In Cappelli, blue light and dichromatic irradiation (blue plus red) gave lower mitotic indices than the dark control, indicating the action of a specific blue-light-absorbing photoreceptor, whereas in Creso the response kinetics to all light regimes which included blue light were more complex. On the basis also of the results obtained with GA₃ application in Cappelli, it appears that (i) the hormonal treatment is able to change the pattern of mitotic index only in the presence of the action of a blue-light receptor and (ii) the different responses of the two cultivars could be the result of different endogenous hormonal levels. The importance of the observations in relation to the data for first-leaf longitudinal growth reported in a previous paper (Baroncelli et al. 1984, Planta 160, 298–304) is discussed.Abbreviations BL blue light - D darkness - FR far-red light - GA gibberellin - GA₃ gibberellic acid - m.i. mitotic index - Norflurazon 4-chloro-5-(methylamino)-2-(,,,-trifluoro-m-totyl-3(2H)) pyridazinone - R red light - WL white light - phytochrome photoequilibrium     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

Decreased GABAB receptor function in the cerebellum and brain stem of hypoxic neonatal rats: Role of glucose,oxygen and epinephrine resuscitation

Background-

Hypoxia during the first week of life can induce neuronal death in vulnerable brain regions usually associated with an impairment of cognitive function that can be detected later in life. The neurobiological changes mediated through neurotransmitters and other signaling molecules associated with neonatal hypoxia are an important aspect in establishing a proper neonatal care.

Methods-

The present study evaluated total GABA, GABA_B receptor alterations, gene expression changes in GABA_B receptor and glutamate decarboxylase in the cerebellum and brain stem of hypoxic neonatal rats and the resuscitation groups with glucose, oxygen and epinephrine. Radiolabelled GABA and baclofen were used for receptor studies of GABA and GABA_B receptors respectively and Real Time PCR analysis using specific probes for GABA_B receptor and GAD mRNA was done for gene expression studies.

Results-

The adaptive response of the body to hypoxic stress resulted in a reduction in total GABA and GABA_B receptors along with decreased GABA_B receptor and GAD gene expression in the cerebellum and brain stem. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations.

Conclusions-

Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation, which helps in encountering hypoxia. The present study suggests that reduction in the GABA_B receptors functional regulation during hypoxia plays an important role in central nervous system damage. Resuscitation with glucose alone and glucose and oxygen to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor

Summary The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the -amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

Further Characterization of 5-HT1A Receptors in the Goldfish Retina: Role of Cyclic AMP in the Regulation of the In Vitro Outgrowth of Retinal Explants

The presence of serotonin 5-HT_1A receptors and their physiological role were further characterized in the goldfish retina. The effects of the 5-HT_6/7 receptor antagonists pimozide, fluphenazine and amoxapine, the 5-HT_1A receptor antagonist WAY-100,135, and the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, on the 5-HT_1A receptor agonist [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes, were evaluated. In addition, the effects of serotonin, 8-hydroxy-2-(di-n-propylamino)tetralin, WAY-100,135, the adenylate cyclase inhibitors SQ22536 and MDL12330A, and the cyclic AMP analog 8-bromoadenosine-3:5 cyclic monophosphate were also studied on neuritic outgrowth from retinal explants. WAY-100,135 but not 5-HT_6/7receptor antagonists inhibited [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding to retinal membranes N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline decreased [³H]8-hydroxy-2-(di-n-propylamino)tetralin binding sites up to 70%, while receptor turnover was similar to that reported in other tissues. Serotonin and 8-hydroxy-2-(di-n-propylamino)tetralin stimulated cyclic AMP production, both ex vivo and in vitro, and these increases were related to inhibition of neuritic outgrowth. The inhibitory effect was reduced by SQ22536 and by WAY-100,135, and was mimicked by 8-bromoadenosine-3:5cyclic monophosphate. This study supports previous findings about the role of serotonin as a regulator of axonal outgrowth during in vitro regeneration of the goldfish retina and demonstrates that this effect is mediated, at least in part, by 5-HT_1A receptors through a mechanism which involves an increase of cyclic AMP levels.     

Clozapine's Antipsychotic Effects do not Depend on Blockade of 5-HT3 Receptors

Sixteen known 5-HT₃ receptor blockers, including clozapine, fully or partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, indicating that they are also GABA_A antagonists, some of them selective for subsets of GABA_A receptors. The 5-HT₃ receptor blocker, ondansetron, has been reported to produce some antipsychotic and anxiolytic effects. However, no antipsychotic effects have been reported for a large number of highly potent 5-HT₃ receptor blockers. Like clozapine, ondansetron partially reverses the inhibitory effect of GABA on [³⁵S]TBPS binding. Additivity experiments suggest that ten 5-HT₃ receptor blockers tested at low concentrations preferentially block subtypes of GABA_A receptors that are among those blocked by clozapine. Wiley and Porter (29) reported that MDL-72222, the most potent GABA_A antagonist decribed here, partially generalizes (71%) with clozapine in rats trained to discriminate an interoceptive clozapine stimulus, but only at a dose that severly decreases responding. Tropisetron (ICS-205,930) exhibits both GABA-positive and GABA-negative effects. R-(+)-zacopride is 6-fold more potent than S-(–)-zacopride as a GABA_A antagonist. We conclude that the observed antipsychotic and, possibly, anxiolytic effects of some 5-HT₃ receptor blockers are due to selective antagonism of certain GABA_A receptors, and not to blockade of 5-HT₃ receptors. We speculate that the anxiolytic and sedative effects of clozapine and several other antipsychotic drugs may be due to selective blockade of 122 GABA_A receptors which are preferentially located on certain types of GABAergic interneurons (probably parvalbumin positive). Blockade of these receptors will increase the inhibitory output of these interneurons. So far, no highly potent GABA_A antagonists with clozapine-like selectivity have been identified. Such compounds may exhibit improved clozapine-like antipsychotic activity.     

Roles of M3 receptor in the effect of penehyclidine hydrochloride upregulated beta-arrestin-1 expression in LPS-stimulated HPMVEC

Background: This study is to investigate the roles of muscarinic receptor 3 (M₃ receptor) in the effect of penehyclidine hydrochloride (PHC) upregulated beta-arrestin-1 expression in lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMVEC).

Methods: HPMVECs were transfected with a shRNA-containing plasmid that specifically targets M₃ receptor mRNA. Cells were collected to measure F-actin contents, levels of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as changes of F-actin cytoskeleton arrangement by Laser scanning confocal. Beta-arrestin-1 protein expressions were determined by Western blot and beta-arrestin-1 mRNA expressions were measured by Real-time PCR.

Results: Similar to normal cells, PHC could also increase F-actin contents and beta-arrestin-1 expressions, reduce ICAM-1 and VCAM-1 expressions, and inhibit LPS-stimulated reorganization of F-actin and formation of stress fiber in M₃ receptor shRNA group. Compared with normal cells, F-actin cytoskeleton was neat, ICAM-1 and VCAM-1 expressions were decreased, as well as F-actin contents were increased in M₃ receptor shRNA group. However, there were no differences in beta-arrestin-1 expressions between normal cell groups and M₃ receptor shRNA groups.

Conclusion: These results indicate that M₃ receptor plays an important role in pulmonary microvascular endothelial barrier function, and knock-out of M₃ receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. However, upregulative effect of PHC on beta-arrestin-1 expression is independent with presence of M₃ receptor.     

GABA-Dopamine Receptor-Receptor Interactions in Neostriatal Membranes of the Rat

Recent evidence has shown in membrane preparations that the binding of one ligand to its receptor is able to modify the binding parameters of a second receptor (receptor-receptor interactions), allowing the modulation of incoming signals onto a neuron. To further understand the -amino-butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experiments to explore whether an activation of the GABA_A receptor could affect the binding characteristics of the D₂ DA receptor in membrane preparations of the rat neostriatum. The results show that GABA (30–100 nM) significantly increases the dissociation constant of the high affinity (K_H) D₂ DA binding site (labelled with the selective D₂ DA receptor antagonist [³H]raclopride and that such an effect is fully counteracted by the GABA_A receptor antagonist bicuculline (1 M). It is suggested that such putative GABA_A/D₂ receptor-receptor interactions may take place in the somato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate the inhibitory effects of DA on these neurons, mediated via D₂ receptors.     

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

Objectives: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to G_q/11 proteins.

Methods: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP).

Results: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of G_q/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of G_iα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of G_iα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling.

Conclusion: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.     

Studies on the Cytosolic Phospholipase A2 in Immortalized Astrocytes (DITNC) Revealed New Properties of the Calcium Ionophore,A23187

Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A₂ (PLA₂) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA₂ has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A₂ in immortalized astrocytes (DITNC). Incubation of DITNC cells with [¹⁴C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4–20 M) and thimerosal (100 M), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 M), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca²⁺ and was inhibited by mepacrine (50 M), a non-specific PLA₂ inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA₂ in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA₂, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.