G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

Mechanisms of inverse agonist action at D-2 dopamine receptors

1 Mechanisms of inverse agonist action at the D-2(short) dopamine receptor have been examined. 2 Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [H-3]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. 3 Competition of inverse agonists versus [H-3] NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K-i values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K-coupled and K-uncoupled were statistically different for the set of compounds tested ( ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. 4 These observations were supported by simulations of these competition experiments according to the extended ternary complex model. 5 Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [S-35]GTPγ S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. 6 These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.

Mechanisms of G protein activation via the D-2 dopamine receptor: evidence for persistent receptor/G protein interaction after agonist stimulation

Background and purpose: The aim of this report is to study mechanisms of G protein activation by agonists. Experimental approach: The association and dissociation of guanosine 5'-O-(3-[S-35] thio) triphosphate ([S-35] GTP gamma S) binding at G proteins in membranes of CHO cells stably transfected with the human dopamine D-2short receptor was studied in the presence of a range of agonists. Key results: Binding of [S-35] GTPgS was dissociable in the absence of agonist and dissociation was accelerated both in rate and extent by dopamine, an effect which was blocked by the dopamine D-2 receptor antagonist raclopride and by suramin, which inhibits receptor/G protein interaction. A range of agonists of varying efficacy increased the rate of dissociation of [S-35] GTPgS binding, with the more efficacious agonists resulting in faster dissociation. Agonists were able to dissociate about 70% of the pre-bound [S-35] GTPgS, leaving a component which may not be accessible to the agonist-bound receptor. The dissociable component of the [S-35] GTPgS binding was reduced with longer association times and increased [S-35] GTPgS concentrations. Conclusions and implications: These data are consistent with [S-35] GTPgS binding being initially to receptor-linked G proteins and then to G proteins which have separated from the agonist bound receptor. Under the conditions used typically for [S-35] GTPgS binding assays, therefore, much of the agonist-receptor complex remains in proximity to G proteins after they have been activated by agonist.

Assays for enhanced activity of low efficacy partial agonists at the D-2 dopamine receptor

Background and purpose: Low efficacy partial agonists at the D-2 dopamine receptor may be useful for treating schizophrenia. In this report we describe a method for assessing the efficacy of these compounds based on stimulation of [S-35]GTP gamma S binding. Experimental approach: Agonist efficacy was assessed from [S-35]GTP gamma S binding to membranes of CHO cells expressing D2 dopamine receptors in buffers with and without Na+. Effects of Na+ on receptor/G protein coupling were assessed using agonist/[H-3] spiperone competition binding assays. Key results: When [S-35]GTP gamma S binding assays were performed in buffers containing Na+, some agonists (aripiprazole, AJ-76, UH-232) exhibited very low efficacy whereas other agonists exhibited measurable efficacy. When Na+ was substituted by N-methyl D-glucamine, the efficacy of all agonists increased (relative to that of dopamine) but particularly for aripiprazole, aplindore, AJ-76, (-)-3-PPP and UH-232. In ligand binding assays, substitution of Na+ by N-methyl D-glucamine increased receptor/G protein coupling for some agonists -. aplindore, dopamine and (-)-3-PPP-but for aripiprazole, AJ-76 and UH-232 there was little effect on receptor/G protein coupling. Conclusions and implications: Substitution of Na+ by NMDG increases sensitivity in [S-35] GTPgS binding assays so that very low efficacy agonists were detected clearly. For some agonists the effect seems to be mediated via enhanced receptor/G protein coupling whereas for others the effect is mediated at another point in the G protein activation cycle. AJ-76, aripiprazole and UH-232 seem particularly sensitive to this change in assay conditions. This work provides a new method to discover these very low efficacy agonists.

Microglial activation correlates with severity in Huntington disease: a clinical and PET study

Background: Huntington disease ( HD) is characterized by the progressive death of medium spiny dopamine receptor bearing striatal GABAergic neurons. In addition, microglial activation in the areas of neuronal loss has recently been described in postmortem studies. Activated microglia are known to release neurotoxic cytokines, and these may contribute to the pathologic process. Methods: To evaluate in vivo the involvement of microglia activation in HD, the authors studied patients at different stages of the disease using [ C-11]( R)-PK11195 PET, a marker of microglia activation, and [ C-11] raclopride PET, a marker of dopamine D2 receptor binding and hence striatal GABAergic cell function. Results: In HD patients, a significant increase in striatal [ C-11]( R)-PK11195 binding was observed, which significantly correlated with disease severity as reflected by the striatal reduction in [ C-11] raclopride binding, the Unified Huntington's Disease Rating Scale score, and the patients' CAG index. Also detected were significant increases in microglia activation in cortical regions including prefrontal cortex and anterior cingulate. Conclusions: These [ C-11]( R)-PK11195 PET findings show that the level of microglial activation correlates with Huntington disease ( HD) severity. They lend support to the view that microglia contribute to the ongoing neuronal degeneration in HD and indicate that [ C-11]( R)-PK11195 PET provides a valuable marker when monitoring the efficacy of putative neuroprotecting agents in this relentlessly progressive genetic disorder.

The relationship between membrane damage, release of protein and loss of viability in Escherichia coli exposed to high hydrostatic pressure

The aim of this work was to examine a possible association between resistance of two Escherichia coli strains to high hydrostatic pressure and the susceptibility of their cell membranes to pressure-induced damage. Cells were exposed to pressures between 100 and 700 MPa at room temperature (~20C) in phosphate-buffered-saline. In the more pressure-sensitive strain E. coli 8164, loss of viability occurred at pressures between 100 MPa and 300 MPa and coincided with irreversible loss of membrane integrity as indicated by uptake of propidium iodide (PI) and leakage of protein of molecular mass between 9 and 78 kDa from the cells. Protein release increased to a maximum at 400 MPa then decreased, possibly due to intracellular aggregation at the higher pressures. In the pressure-resistant strain E. coli J1, PI was taken up during pressure treatment but not after decompression indicating that cells were able to reseal their membranes. Loss of viability in strain J1 coincided with the transient loss of membrane integrity between approximately 200 MPa and 600 MPa. In E. coli J1 leakage of protein occurred before loss of viability and the released protein was of low molecular mass, between 8 and 11 kDa and may have been of periplasmic origin. In these two strains differences in pressure resistance appeared to be related to differences in the ability of their membranes to withstand disruption by pressure. However it appears that transient loss of membrane integrity during pressure can lead to cell death irrespective of whether cells can reseal their membranes afterwards.

PEGylated amyloid peptide nanocontainer delivery and release system

A micellar nanocontainer delivery and release system is designed on the basis of a peptide-polymer conjugate. The hybrid molecules self-assemble into micelles comprising a modified amyloid peptide core surrounded by a PEG corona. The modified amyloid peptide previously studied in our group forms helical ribbons based on a beta-sheet motif and contains beta-amino acids that are excluded from the beta-sheet structure, thus being potentially useful as fibrillization inhibitors. In the model peptide-PEG hybrid system studied, enzymatic degradation using alpha-chymotrypsin leads to selective cleavage close to the PEG-peptide linkage, break up of the micelles, and release of peptides in unassociated form. The release of monomeric peptide is useful because aggregation of the released peptide into beta-sheet amyloid fibrils is not observed. This concept has considerable potential in the targeted delivery of peptides for therapeutic applications.

Polymorphisms in dopamine system genes are associated with individual differences in attention in infancy

Knowledge about the functional status of the frontal cortex in infancy is limited. This study investigated the effects of polymorphisms in four dopamine system genes on performance in a task developed to assess such functioning, the Freeze-Frame task, at 9 months of age. Polymorphisms in the catechol-O-methyltransferase (COMT) and the dopamine D4 receptor (DRD4) genes are likely to impact directly on the functioning of the frontal cortex, whereas polymorphisms in the dopamine D2 receptor (DRD2) and dopamine transporter (DAT1) genes might influence frontal cortex functioning indirectly via strong frontostriatal connections. A significant effect of the COMT valine158methionine (Val158Met) polymorphism was found. Infants with the Met/Met genotype were significantly less distractible than infants with the Val/Val genotype in Freeze-Frame trials presenting an engaging central stimulus. In addition, there was an interaction with the DAT1 3′ variable number of tandem repeats polymorphism; the COMT effect was present only in infants who did not have two copies of the DAT1 10-repeat allele. These findings indicate that dopaminergic polymorphisms affect selective aspects of attention as early as infancy and further validate the Freeze-Frame task as a frontal cortex task.

Constraint release in entangled binary blends of linear polymers: a molecular dynamics study

We present extensive molecular dynamics simulations of the dynamics of diluted long probe chains entangled with a matrix of shorter chains. The chain lengths of both components are above the entanglement strand length, and the ratio of their lengths is varied over a wide range to cover the crossover from the chain reptation regime to tube Rouse motion regime of the long probe chains. Reducing the matrix chain length results in a faster decay of the dynamic structure factor of the probe chains, in good agreement with recent neutron spin echo experiments. The diffusion of the long chains, measured by the mean square displacements of the monomers and the centers of mass of the chains, demonstrates a systematic speed-up relative to the pure reptation behavior expected for monodisperse melts of sufficiently long polymers. On the other hand, the diffusion of the matrix chains is only weakly perturbed by the diluted long probe chains. The simulation results are qualitatively consistent with the theoretical predictions based on constraint release Rouse model, but a detailed comparison reveals the existence of a broad distribution of the disentanglement rates, which is partly confirmed by an analysis of the packing and diffusion of the matrix chains in the tube region of the probe chains. A coarse-grained simulation model based on the tube Rouse motion model with incorporation of the probability distribution of the tube segment jump rates is developed and shows results qualitatively consistent with the fine scale molecular dynamics simulations. However, we observe a breakdown in the tube Rouse model when the short chain length is decreased to around N-S = 80, which is roughly 3.5 times the entanglement spacing N-e(P) = 23. The location of this transition may be sensitive to the chain bending potential used in our simulations.

Platelets release novel thiol isomerase enzymes which are recruited to the cell surface following activation

The thiol isomerase enzymes protein disulphide isomerase (PDI) and endoplasmic reticulum protein 5 (ERp5) are released by resting and activated platelets. These re-associate with the cell surface where they modulate a range of platelet responses including adhesion, secretion and aggregation. Recent studies suggest the existence of yet uncharacterised platelet thiol isomerase proteins. This study aimed to identify which other thiol isomerase enzymes are present in human platelets. Through the use of immunoblotting, flow cytometry, cell-surface biotinylation and gene array analysis, we report the presence of five additional thiol isomerases in human and mouse platelets and megakaryocytes, namely; ERp57, ERp72, ERp44, ERp29 and TMX3. ERp72, ERp57, ERp44 and ERp29 are released by platelets and relocate to the cell surface following platelet activation. The transmembrane thiol isomerase TMX3 was also detected on the platelet surface but does not increase following activation. Extracellular PDI is also implicated in the regulation of coagulation by the modulation of tissue factor activity. ERp57 was identified within platelet-derived microparticle fractions, suggesting that ERp57 may also be involved in the regulation of coagulation as well as platelet function. These data collectively implicate the expanding family of platelet-surface thiol isomerases in the regulation of haemostasis.

Sulforaphane protects cortical neurons against 5-S-cysteinyl-dopamine-induced toxicity through the activation of ERK1/2, Nrf-2 and the upregulation of detoxification enzymes

The degeneration of dopaminergic neurons in the substantia nigra has been linked to the formation of the endogenous neurotoxin 5-S-cysteinyl-dopamine. Sulforaphane (SFN), an isothiocyanate derived from the corresponding precursor glucosinolate found in cruciferous vegetables has been observed to exert a range of biological activities in various cell populations. In this study, we show that SFN protects primary cortical neurons against 5-S-cysteinyl-dopamine induced neuronal injury. Pre-treatment of cortical neurons with SFN (0.01-1 microM) resulted in protection against 5-S-cysteinyl-dopamine-induced neurotoxicity, which peaked at 100 nM. This protection was observed to be mediated by the ability of SFN to modulate the extracellular signal-regulated kinase 1 and 2 and the activation of Kelch-like ECH-associated protein 1/NF-E2-related factor-2 leading to the increased expression and activity of glutathione-S-transferase (M1, M3 and M5), glutathione reductase, thioredoxin reductase and NAD(P)H oxidoreductase 1. These data suggest that SFN stimulates the NF-E2-related factor-2 pathway of antioxidant gene expression in neurons and may protect against neuronal injury relevant to the aetiology of Parkinson's disease.

Caffeic acid, tyrosol and p-coumaric acid are potent inhibitors of 5-S-cysteinyl-dopamine induced neurotoxicity

Parkinson's disease is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra. Recent investigations have shown that conjugates such as the 5-S-cysteinyl-dopamine, possess strong neurotoxicity and may contribute to the underlying progression of the disease pathology. Although the neuroprotective actions of flavonoids are well reported, that of hydroxycinnamates and other phenolic acids is less established. We show that the hydroxycinnamates caffeic acid and p-coumaric acid, the hydroxyphenethyl alcohol, tyrosol, and a Champagne wine extract rich in these components protect neurons against injury induced by 5-S-cysteinyl-dopamine in vitro. The protection induced by these polyphenols was equal to or greater than that observed for the flavonoids, (+)-catechin, (-)-epicatechin and quercetin. For example, p-coumaric acid evoked significantly more protection at 1muM (64.0+/-3.1%) than both (-)-epicatechin (46.0+/-4.1%, p<0.05) and (+)-catechin (13.1+/-3.0%, p<0.001) at the same concentration. These data indicate that hydroxycinnamates, phenolic acids and phenolic alcohol are also capable of inducing neuroprotective effects to a similar extent to that seen with flavonoids.

The effect of the year of wheat variety release on productivity and stability of performance on two organic and two non-organic farms

Nineteen wheat cultivars, released from 1934 to 2000, were grown at two organic and two non-organic sites in each of 3 years. Assessments included grain yield, grain protein concentration, protein yield, disease incidence and green leaf area. The superiority of each cultivar (the sum of the squares of the differences between its mean in each environment and the mean of the best cultivar there, divided by twice the number of environments; CS) was calculated for yield, grain protein concentration and protein yield, and ranked in each environment. The yield and grain protein concentration CS were more closely correlated with cultivar release date at the non-organic sites than at organic sites. This difference may be attributed to higher yield levels with larger differences among cultivars at the non-organic sites, rather than to improved stability (i.e. similar ranks) across sites. The significant difference in the correlation of protein yield CS and cultivar age between organic and non-organic sites would support evidence that the ability to take up mineral nitrogen (N) compared to soil N has been a component of the selection conditions of more modern cultivars (released after 1989). This is supported by assessment of green leaf area (GLA), where more modern cultivars in the non-organic systems had greater late-season GLA, a trend that was not identified in organic conditions. This effect could explain the poor correlation between age and protein yield CS in organic compared to non-organic conditions where modern cultivars are selected to benefit from later nitrogen (N) availability which includes the spring nitrogen applications tailored to coincide with peak crop demand. Under organic management, N release is largely based on the breakdown of fertility-building crops incorporated (ploughed-in) in the previous autumn. The release of nutrients from these residues is dependent on the soil conditions, which includes temperature and microbial populations, in addition to the potential leaching effect of high winter rainfall in the UK. In organic cereal crops, early resource capture is a major advantage for maximizing the utilization of nutrients from residue breakdown. It is concluded that selection of cultivars under conditions of high agrochemical inputs selects for cultivars that yield well under maximal conditions in terms of nutrient availability and pest, disease and weed control. The selection conditions for breeding have a tendency to select cultivars which perform relatively better in non-organic compared to organic systems.

Second meal effect: modified sham feeding does not provoke the release of stored triacylglycerol from a previous high-fat meal

The present study was carried out to determine whether cephalic stimulation, associated with eating a meal, was sufficient stimulus to provoke the release of stored triacylglycerol (TAG) from a previous high-fat meal. Ten subjects were studied on three separate occasions. Following a 12 h overnight fast, subjects were given a standard mixed test meal which contained 56 g fat. Blood samples were taken before the meal and for 5 h after the meal when the subjects were randomly allocated to receive either water (control) or were modified sham fed a low-fat (6 g fat) or moderate-fat (38 g fat) meal. Blood samples were collected for a further 3 h. Compared with the control, modified sham feeding a low- or moderate-fat meal did not provoke an early entry of TAG, analysed in either plasma or TAG-rich lipoprotein (TRL) fraction (density ,1´006 kg/l). The TRL-retinyl ester data showed similar findings. A cephalic phase secretion of pancreatic polypeptide, without a significant increase in cholecystokinin levels, was observed on modified sham feeding. Although these data indicate that modified sham feeding was carried out successfully, analysis of the fat content of the expectorant showed that our subjects may have accidentally ingested a small amount of fat (0´7 g for the low-fat meal and 2´4 g for the moderate-fat meal). Nevertheless, an early TAG peak following modified sham feeding was not demonstrated in the present study, suggesting that significant ingestion of food, and not just orosensory stimulation, is necessary to provoke the release of any TAG stored from a previous meal.