Interaction studies of multiple binding sites on m4 muscarinic acetylcholine receptors. (25/122)

This study investigated the reciprocal cross-interactions between two distinct allosteric sites on the M(4) muscarinic acetylcholine receptor (mAChR) in the absence or presence of different orthosteric ligands. Initial studies revealed that two novel benzimidazole allosteric modulators, 17-beta-hydroxy-17-alpha-ethy nyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole (WIN 62,577) and 17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimid azole (WIN 51,708), exhibited different degrees of positive, negative, or close-to-neutral cooperativity with the orthosteric site on M(1) or M(4) mAChRs, depending on the chemical nature of the orthosteric radioligand that was used [[(3)H]N-methylscopolamine ([(3)H]NMS) versus [(3)H]quinuclidinylbenzilate ([(3)H]QNB)]. The largest window for observing an effect (negative cooperativity) was noted for the combination of WIN 62,577 and [(3)H]QNB at the M(4) mAChR. Experiments involving the combination of these two ligands with unlabeled agonists [acetylcholine, 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium (McN-A-343), or xanomeline] revealed low degrees of negative cooperativity between WIN 62,577 and each agonist, whereas stronger negative cooperativity was observed against atropine. It is interesting that when these experiments were repeated using the prototypical modulators heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3-phth), alcuronium, or brucine (which act at a separate allosteric site), WIN 62,577 exhibited negative cooperativity with each modulator when the orthosteric site was unoccupied, but this switched to neutral cooperativity when the receptor was occupied by [(3)H]QNB. Dissociation kinetic experiments using [(3)H]NMS and combination of C(7)/3-phth with WIN 62,577 also provided evidence for neutral cooperativity between the two allosteric sites when the orthosteric site is occupied. Together, these results provide insight into the nature of the interaction between two distinct allosteric sites on the M(4) mAChR and how this interaction is perturbed upon occupancy of the orthosteric site.  (+info)

The altered specificity of cortisone reductase with certain retroandrostan-3-one substrates. (26/122)

The retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione were good substrates for cortisone reductase in the presence of NADH, and the products corresponded to the respective 3beta-hydroxy compounds, in which the 3beta-hydroxyl group is axial and the absolute configuration is 3S. The analogous natural steroids 17beta-hydroxy-5beta,9alpha,10beta-androstan-3-one and 5beta,9alpha,10beta-androstane-3,17-dione were very poor substrates, and gave the corresponding 3alpha(equatorial,3R)-hydroxy compounds, and, in the latter case, also an appreciable amount of 3beta(axial, 3S)-hydroxy-5beta,9alpha,10beta-androstan-17-one. The natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione were better substrates than the retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one, but were not such good substrates as the retro steroids 17beta-hydroxy-5beta,9beta,10alpha-androstan-3-one and 5beta,9beta,10alpha-androstane-3,17-dione. Unlike these retro steroid 5beta,9beta,10alpha-androstan-3-ones, the natural steroids 17beta-hydroxy-5alpha,9alpha,10beta-androstan-3-one and 5alpha,9alpha,10beta-androstane-3,17-dione gave the corresponding 3alpha(axial,3R)-hydroxy compounds. The retro steroid 17beta-hydroxy-5alpha,9beta,10alpha-androstan-3-one was not a good substrate, and the product of reaction corresponded to the 3alpha(axial,3R)-hydroxy compound. The nature of substrate recognition by this enzyme is discussed in the light of these structure-activity relationships.  (+info)

Coexisting stripe- and patch-shaped domains in giant unilamellar vesicles. (27/122)

We report a new type of gel-liquid phase segregation in giant unilamellar vesicles (GUVs) of mixed lipids. Coexisting patch- and stripe-shaped gel domains in GUV bilayers composed of DOPC/DPPC or DLPC/DPPC are observed by confocal fluorescence microscopy. The lipids in stripe domains are shown to be tilted according to the DiIC18 fluorescence intensity dependence on the excitation polarization. The patch domains are found to be mainly composed of DPPC-d62 according to the coherent anti-Stokes Raman scattering (CARS) images of DOPC/DPPC-d62 bilayers. When cooling GUVs from above the miscibility temperature, the patch domains start to appear between the chain melting and the pretransition temperature of DPPC. In GUVs containing a high molar percentage of DPPC, the stripe domains form below the pretransition temperature. Our observations suggest that the patch and stripe domains are in the Pbeta' and Lbeta' gel phases, respectively. According to the thermoelastic properties of GUVs described by Needham and Evans [(1988) Biochemistry 27, 8261-8269], the Pbeta' and Lbeta' phases are formed at relatively low and high membrane tensions, respectively. GUVs with high DPPC percentage have high membrane surface tension and thus mainly exhibit Lbeta' domains, while GUVs with low DPPC percentage have low membrane surface tension and form Pbeta' domains accordingly. Adding negatively charged lipid to the lipid mixtures or applying an osmotic pressure to GUVs using sucrose solutions releases the surface tension and leads to the disappearance of the Lbeta' gel phase. The relationship between the observed domains in free-standing GUV bilayers and those in supported bilayers is discussed.  (+info)

Co-expression of human ABCG5 and ABCG8 in insect cells generates an androstan stimulated membrane ATPase activity. (28/122)

Mutations in the ATP-binding cassette (ABC) proteins ABCG5 or ABCG8 cause sitosterolemia, a condition with increased accumulation of plant sterols. Upon high level expression of the ABCG5 and ABCG8 proteins in baculovirus-Sf9 cell expression system we found a distinct, vanadate sensitive ATPase activity in isolated membrane preparations only when the two proteins were co-expressed. This ATPase activity was significantly stimulated by the addition of certain androgen hormones and analogs, and was effectively inhibited by progesterone. Our results provide a new aspect of biochemical and functional characterization of the ABCG5/ABCG8 proteins and their possible involvement in steroid hormone transport or regulation.  (+info)

Solubility versus electrostatics: what determines lipid/protein interaction in lung surfactant. (29/122)

Mammalian lung surfactant is a complex lipid/protein mixture covering the alveolar interface and has the crucial function of reducing the surface tension at this boundary to minimal values. Surfactant protein SP-B plays an important role for this purpose and was the focus of many recent studies. However, the specificity of lipid/SP-B interactions is controversial. Since these investigations were accomplished at varying pH conditions (pH 5.5 and 7.0), we studied the specificity of these interactions in a dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylglycerol (DPPG)/SP-B (4:1:0.2 mol %) model system at either pH. Mainly fluorescence microscopy and laterally resolved time-of-flight secondary ion mass spectrometry were used to reveal information about the phase behavior of the lipids and the molecular distribution of SP-B in the lipid mixture. DPPG forms separated condensed domains due to a strong hydrogen-bond network, from which the protein is mainly excluded. Considering the protein as an impurity of the lipid mixture leads to the principle of the zone melting process: an impurity is highly more soluble in a liquid phase than in a solid phase. The phase behavior effect of the lipids mainly outperforms the electrostatic interactions between DPPG and SP-B, leading to a more passively achieved colocalization of DPPC and SP-B.  (+info)

A 17beta-derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABA A receptors with nanomolar affinity. (30/122)

BACKGROUND AND PURPOSE: High-affinity, subtype-selective antagonists of the neurosteroid binding sites of GABA(A) receptors are not available. We have characterized an allopregnanolone derivative as an antagonist of cerebellar GABA(A) receptors with nanomolar affinity. EXPERIMENTAL APPROACH: Receptor binding and electrophysiological methods were used for the allosteric modulation of cerebellar GABA(A) receptors by an allopregnanolone derivative, (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3alpha-ol (HBAO). GABA(A) receptors of rat cerebellar membranes were labelled with the chloride channel blocker [(3)H]ethynylbicycloorthobenzoate (EBOB). The ionophore function of GABA(A) receptors was studied by whole-cell patch clamp electrophysiology in cultured rat cerebellar granule and cortical cells. KEY RESULTS: Partial displacement of cerebellar [(3)H]EBOB binding by nanomolar HBAO was attenuated by 0.1 mM furosemide, an antagonist of alpha(6) and beta(2-3) subunit-containing GABA(A) receptors. Displacement curves of HBAO were reshaped by 30 nM GABA and shifted to the right. However, the micromolar potency of full displacement by allopregnanolone was not affected by 0.1 mM furosemide or 30 nM GABA. The nanomolar, but not the micromolar phase of displacement of [(3)H]EBOB binding by GABA was attenuated by 100 nM HBAO. Submicromolar HBAO did not affect [(3)H]EBOB binding to cortical and hippocampal GABA(A) receptors. HBAO up to 1 microM did not affect chloride currents elicited by 0.3-10 microM GABA, while it abolished potentiation by 1 microM allopregnanolone with nanomolar potency in cerebellar but not in cortical cells. Furosemide attenuated cerebellar inhibition by 100 nM HBAO. CONCLUSIONS AND IMPLICATIONS: HBAO is a selective antagonist of allopregnanolone, a major endogenous positive modulator via neurosteroid sites of cerebellar (probably alpha(6)beta(2-3)delta) GABA(A) receptors.  (+info)

Aryl-hydrocarbon receptor activation regulates constitutive androstane receptor levels in murine and human liver. (31/122)

The aryl-hydrocarbon receptor (AhR) is a basic helix-loop-helix/Per-Arnt-Sim transcription factor that can be activated by exogenous as well as endogenous ligands. AhR is traditionally associated with xenobiotic metabolism. In an attempt to identify novel target genes, C57BL/6J mice were treated with beta-naphthoflavone (BNF), a known AhR ligand, and genome-wide expression analysis studies were performed using high-density microarrays. Constitutive androstane receptor (CAR) was found to be one of the differentially regulated genes. Real-time quantitative polymerase chain reaction (qPCR) verified the increase in CAR messenger RNA (mRNA) level. BNF treatment did not increase CAR mRNA in AhR-null mice. Time-course studies in mice revealed that the regulation of CAR mRNA mimicked that of Cyp1A1, a known AhR target gene. To demonstrate that the increase in CAR mRNA translates to an increase in functional CAR protein, mice were sequentially treated with BNF (6 hours) followed by the selective CAR agonist, TCPOBOP (3 hours). qPCR revealed an increase in the mRNA level of Cyp2b10, previously known to be regulated by CAR. This also suggests that CAR protein is present in limiting amounts with respect to its transactivation ability. Finally, CAR was also up-regulated in primary human hepatocytes in response to AhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene. CONCLUSION: This study identifies a mode of up-regulating CAR and potentially expands the role of AhR in drug metabolism. This study also demonstrates in vivo up-regulation of CAR through chemical exposure.  (+info)

Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins). (32/122)

Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.  (+info)