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At least 991 records · Page 55Linked to original sources
PubMed2001

An inverse dynamics approach to face animation.

Muscle-based models of the human face produce high quality animation but rely on recorded muscle activity signals or synthetic muscle signals that are often derived by trial and error. This paper presents a dynamic inversion of a muscle-based model (Lucero and Munhall, 1999) that permits the animation to be created from kinematic recordings of facial movements. Using a nonlinear optimizer (Powell's algorithm), the inversion produces a muscle activity set for seven muscles in the lower face that minimize the root mean square error between kinematic data recorded with OPTOTRAK and the corresponding nodes of the modeled facial mesh. This inverted muscle activity is then used to animate the facial model. In three tests of the inversion, strong correlations were observed for kinematics produced from synthetic muscle activity, for OPTOTRAK kinematics recorded from a talker for whom the facial model is morphologically adapted and finally for another talker with the model morphology adapted to a different individual. The correspondence between the animation kinematics and the three-dimensional OPTOTRAK data are very good and the animation is of high quality. Because the kinematic to electromyography (EMG) inversion is ill posed, there is no relation between the actual EMG and the inverted EMG. The overall redundancy of the motor system means that many different EMG patterns can produce the same kinematic output.

Adult↗
PubMed2002

Quantifying uncertainty in geoacoustic inversion. II. Application to broadband, shallow-water data.

This paper applies the new method of fast Gibbs sampling (FGS) to estimate the uncertainties of seabed geoacoustic parameters in a broadband, shallow-water acoustic survey, with the goal of interpreting the survey results and validating the method for experimental data. FGS applies a Bayesian approach to geoacoustic inversion based on sampling the posterior probability density to estimate marginal probability distributions and parameter covariances. This requires knowledge of the statistical distribution of the data errors, including both measurement and theory errors, which is generally not available. Invoking the simplifying assumption of independent, identically distributed Gaussian errors allows a maximum-likelihood estimate of the data variance and leads to a practical inversion algorithm. However, it is necessary to validate these assumptions, i.e., to verify that the parameter uncertainties obtained represent meaningful estimates. To this end, FGS is applied to a geoacoustic experiment carried out at a site off the west coast of Italy where previous acoustic and geophysical studies have been performed. The parameter uncertainties estimated via FGS are validated by comparison with: (i) the variability in the results of inverting multiple independent data sets collected during the experiment; (ii) the results of FGS inversion of synthetic test cases designed to simulate the experiment and data errors; and (iii) the available geophysical ground truth. Comparisons are carried out for a number of different source bandwidths, ranges, and levels of prior information, and indicate that FGS provides reliable and stable uncertainty estimates for the geoacoustic inverse problem.

Acoustics↗
PubMed2002

Inverse calculation of material parameters for a thin-layer system using transient elastic waves.

The inverse calculation of material parameters of a thin-layer system is investigated using transient elastic waves. The inverse problem is formulated as an optimization problem in which the norm of the discrepancies between the calculated and measured normal surface displacements is minimized through the simplex algorithm. The theoretical result is first solved using the Laplace transform and the transient response is then implemented analytically by Cagniard's method. In the experiment, the source time function is generated by the brittle fracture of a pencil lead on the surface of the thin-layer system, and a National Bureau of Standards (NBS) conical transducer is used to record the surface responses. To obtain reliable inverse results for material parameters, a two-step inverse calculation procedure is proposed. The recovered material parameters of the specimens agree well with the theoretical values and experimental results.

Journal Article↗
PubMed2002

An inversion for Biot parameters in water-saturated sand.

The discrepancy between acoustic measurements and the theoretical predictions was investigated in the case of water-saturated sand. Two theoretical models were considered: visco-elastic and poro-elastic solid models. The visco-elastic solid model could not be reconciled with reflection loss measurements and was rejected. The poro-elastic solid model using Biot's theory [J. Acoust. Soc. Am. 103, 2723-2729 (1998)] as formulated by Stoll [J. Acoust. Soc. Am. 70, 149-156 (1981)] was an improvement. It was investigated using an inversion process. Operative values of grain bulk modulus and the frame bulk and shear moduli of water-saturated sand were inverted from simple measurements--reflection loss, compressional and shear wave speeds and attenuations. Although the inversion process is nonlinear, in practice, it is well behaved and converges quite rapidly to a unique solution. The issue of imprecisely known parameter values was handled in a probabilistic manner. The inversion results, using published laboratory and in situ measurements, showed that further improvement was needed. In an attempt to find a solution, two possible hypotheses are put forward. (1) Composite materials: The possibility that the frame may contain fluid and that the pore fluid may contain loose grains. (2) Independent coefficient of fluid content: The possibility that porosity may change with pore fluid pressure. Inversion results were encouraging for both hypotheses. It is difficult to say which of the two hypotheses is superior, and the two hypotheses are not mutually exclusive. The new hypotheses represent a significant advance because they have the potential to resolve the remaining discrepancies. At this stage, alternative interpretations of the data are possible.

Acoustics↗
PubMed2004

Adjoint modeling for acoustic inversion.

The use of adjoint modeling for acoustic inversion is investigated. An adjoint model is derived from a linearized forward propagation model to propagate data-model misfit at the observation points back through the medium to the medium perturbations not being accounted for in the model. This adjoint model can be used to aid in inverting for these unaccounted medium perturbations. Adjoint methods are being applied to a variety of inversion problems, but have not drawn much attention from the underwater acoustic community. This paper presents an application of adjoint methods to acoustic inversion. Inversions are demonstrated in simulation for both range-independent and range-dependent sound speed profiles using the adjoint of a parabolic equation model. Sensitivity and error analyses are discussed showing how the adjoint model enables calculations to be performed in the space of observations, rather than the often much larger space of model parameters. Using an adjoint model enables directions of steepest descent in the model parameters (what we invert for) to be calculated using far fewer modeling runs than if a forward model only were used.

Journal Article↗
PubMed2005

Geoacoustic inversion in time domain using ship of opportunity noise recorded on a horizontal towed array.

A time domain geoacoustic inversion method using ship noise received on a towed horizontal array is presented. The received signal, containing ship noise as a source of opportunity, is time-reversed and then back-propagated to the vicinity of the ship. The back-propagated signal is correlated with the modeled signal which is expected to peak at the ship's location in case of a good match for the environment. This match is utilized for the geoacoustic parameter inversion. The objective function for this optimization problem is thus defined as the normalized power focused in an area around the source position, using a matched impulse response filter. A hybrid use of global and local search algorithms, i.e., GA and Powell's method is applied to the optimization problem. Applications of the proposed inversion method to MAPEX 2000 noise experiment conducted north of the island of Elba show promising results, and it is shown that the time domain inversion takes advantage of dominant frequencies of the source signature automatically.

Journal Article↗
PubMed2005

A robust spatial filtering technique for multisource localization and geoacoustic inversion.

Geoacoustic inversion and source localization using beamformed data from a ship of opportunity has been demonstrated with a bottom-mounted array. An alternative approach, which lies within a class referred to as spatial filtering, transforms element level data into beam data, applies a bearing filter, and transforms back to element level data prior to performing inversions. Automation of this filtering approach is facilitated for broadband applications by restricting the inverse transform to the degrees of freedom of the array, i.e., the effective number of elements, for frequencies near or below the design frequency. A procedure is described for nonuniformly spaced elements that guarantees filter stability well above the design frequency. Monitoring energy conservation with respect to filter output confirms filter stability. Filter performance with both uniformly spaced and nonuniformly spaced array elements is discussed. Vertical (range and depth) and horizontal (range and bearing) ambiguity surfaces are constructed to examine filter performance. Examples that demonstrate this filtering technique with both synthetic data and real data are presented along with comparisons to inversion results using beamformed data. Examinations of cost functions calculated within a simulated annealing algorithm reveal the efficacy of the approach.

Journal Article↗
PubMed2000

Matched-field processing, geoacoustic inversion, and source signature recovery of blue whale vocalizations.

Matched-field processing (MFP) and global inversion techniques have been applied to vocalizations from four whales recorded on a 48-element tilted vertical array off the Channel Islands in 1996. Global inversions from selected whale calls using as few as eight elements extracted information about the surrounding ocean bottom composition, array shape, and the animal's position. These inversion results were then used to conduct straightforward MFP on other calls. The sediment sound-speed inversion estimates are consistent with those derived from sediment samples collected in the area. In general, most animals swam from the east to west, but one animal remained within approximately 500 m of its original position over 45 min. All whales vocalized between 10 and 40 m depth. Three acoustic sequences are discussed in detail: the first illustrating a match between an acoustic track and visual sighting, the second tracking two whales to ranges out to 8 km, and the final sequence demonstrating high-resolution dive profiles from an animal that changed its course to avoid the research platform FLIP (floating instrument platform). This last whale displayed an unusual diversity of signals that include three strong frequency-modulated (FM) downsweeps which contain possible signs of an internal resonance. The arrival of this same whale coincided with a sudden change in oceanographic conditions.

Acoustics↗
PubMed2000

Hybrid geoacoustic inversion of broadband Mediterranean Sea data

This paper describes an acoustic experiment (PROSIM'97) carried out to investigate inversion for seabed properties at a site off the west coast of Italy where previous acoustic and geophysical studies have been performed. Acoustic fields were measured at a vertical hydrophone array due to a swept-frequency source towed over weakly range-dependent bathymetry. Based on the known geology, the seabed is modeled as a sediment layer overlying a semi-infinite basement with unknown model parameters consisting of the sediment thickness, sediment and basement sound speeds, source range and depth, water depth at the source and array, and array tilt. A hybrid inversion algorithm is applied to determine the model values that minimize the mismatch with the measured acoustic fields. Multiple data sets are analyzed to examine the consistency of the inversion results. It is found that the low sound speed of the sediment layer, together with a large uncertainty in bathymetry, leads to strong correlations between the water depths and sediment thickness. This precludes reliable estimation of these parameters individually; however, the total depth to the basement can be estimated reliably. In addition, the basement speed and geometric parameters are estimated consistently, and all parameters compare favorably with the geophysical ground-truth information and with previous inversion results.

Journal Article↗
PubMed2000

Experimental investigation of the pulse inversion technique for imaging ultrasound contrast agents.

The application of ultrasound contrast agents aims to detect low velocity blood flow in the microcirculation. To enhance discrimination between tissue and blood containing the contrast agent, harmonic imaging is used. Harmonic imaging requires the application of narrow-band signals and is obscured by high levels of native harmonics generated in an intervening medium. To improve discrimination between contrast agent and native harmonics, a pulse inversion technique has been proposed. Pulse inversion allows wide-band signals, thus preserving the axial resolution. The present study examines the interference of native harmonics and discusses the practical difficulties of wide-band pulse inversion measurements of harmonics by a single transducer. Native harmonics are not eliminated by pulse inversion. Furthermore, only even harmonics remain and are amplified by 6 dB, alleviating the requirement for selective filtering. Finally, it is shown that the contaminating third harmonic contained in the square wave activation signal leaks through in the emitted signal. The spectral location of the contaminating third harmonic is governed by the transducer spectral characteristics while the location of the native and contrast agent second harmonics is not. Thus the contaminating third harmonic and the native and contrast agent second harmonics may overlap and interfere. Optimal discrimination requires a balance between maximal sensitivity for the second harmonic at reception and minimal interference from the contaminating third harmonic.

Acoustics↗
PubMed2002

Chronic agonist treatment converts antagonists into inverse agonists at delta-opioid receptors.

In cellular models, chronic exposure to mu-opioid agonists converts antagonists into inverse agonists at mu-receptors. Such adaptations could contribute to the development of tolerance and/or dependence. To determine whether delta-receptors respond similarly, or whether this adaptation is unique for mu-receptors, this study examined the effects of prolonged agonist exposure on the intrinsic activity of several delta-opioid ligands in GH(3) cells expressing delta-receptors. In opioid naive cells, delta-receptors were constitutively active, and a series of delta-ligands displayed a range of intrinsic activities for G protein activation. Chronic treatment with the full delta-agonist [D-Pen(2,5)]-enkephalin reduced the acute ability of [D-Pen(2,5)]-enkephalin to stimulate and the full inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174864) to inhibit G protein activation. In contrast, although naloxone and naltriben exhibited weak partial agonism in opioid naive cells, both ligands acted as full inverse agonists to produce concentration-dependent inhibition of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding after prolonged exposure to [D-Pen(2,5)]-enkephalin or to the partial agonist morphine. This effect was reversed by a neutral delta-antagonist (N,N-bisallyl)-Tyr-Gly-Gly-psi-(CH(2)S)-Phe-Leu-OH (ICI-154129). Finally, as is also characteristic of inverse agonists, naloxone and naltriben demonstrated higher affinities for uncoupled delta-receptors in cells chronically treated with [D-Pen(2,5)]-enkephalin, relative to opioid naive cells. Therefore, this relatively novel adaptation is shared by both mu- and delta-opioid receptors and therefore may serve as an important common mechanism involved the development of tolerance and/or dependence.

Adenylyl Cyclases↗
PubMed2004

Inverse agonism and neutral antagonism at wild-type and constitutively active mutant delta opioid receptors.

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity. Here, we have used constitutively active mutant (CAM) delta receptors in two different functional assays, guanosine 5'-O-(3-thio)triphosphate binding and a reporter gene assay, to test potential inverse agonism of 15 delta opioid compounds, originally described as antagonists. These include the classical antagonists naloxone, naltrindole, 7-benzylidene-naltrexone, and naltriben, a new set of naltrindole derivatives, H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-TicPsi[CH2N]Cha-Phe-OH [TICP(Psi)], as well as three 2',6'-dimethyltyrosine-1,2,3,4-tetrahydroquinoline-3-carboxylate (Dmt-Tic) peptides. A reference agonist, SNC 80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], and inverse agonist, ICI 174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu), were also included. In a screen using wild-type and CAM M262T delta receptors, naltrindole (NTI) and close derivatives were mostly inactive, and TIPP behaved as an agonist, whereas Dmt-Tic-OH and N,N(CH3)2-Dmt-Tic-NH2 showed inverse agonism. The two latter compounds showed negative activity across 27 CAM receptors, suggesting that this activity was independent from the activation mechanism. These two compounds also exhibited nanomolar potencies in dose-response experiments performed on wild-type, M262T, Y308H, and C328R CAM receptors. TICP(Psi) exhibited strong inverse agonism at the Y308H receptor. We conclude that the stable N,N(CH3)2-Dmt-Tic-NH2 compound represents a useful tool to explore the spontaneous activity of delta receptors, and NTI and novel derivatives behave as neutral antagonists.

Alkaline Phosphatase↗
PubMed2006

Pharmacological and behavioral profile of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel 5-hydroxytryptamine(2A) receptor inverse agonist.

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of [(3)H]ketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.

Animals↗
PubMed2006

A single point mutation (N514Y) in the human M3 muscarinic acetylcholine receptor reveals differences in the properties of antagonists: evidence for differential inverse agonism.

A single asparagine-to-tyrosine point mutation in the human M muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (approximately 300%) in agonist-independent [3H]inositol phosphate ([3H]IPx) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M3 and (N514Y)M3 mACh receptor-mediated basal [3H]IPx accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the (N514Y)M3 mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate (N514Y)M3 mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the (N514Y)M3 mACh receptor was approximately 100% greater than that seen for the WT-M3 receptor. The ability of antagonists to decrease basal (N514Y)M3 mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [3H]IPx accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.

Cell Line↗
PubMed2006

ZM241385, DPCPX, MRS1706 are inverse agonists with different relative intrinsic efficacies on constitutively active mutants of the human adenosine A2B receptor.

The human adenosine A(2B) receptor belongs to class A G protein-coupled receptors (GPCRs). In our previous work, constitutively active mutant (CAM) human adenosine A(2B) receptors were identified from a random mutation bank. In the current study, three known A(2B) receptor antagonists, 4-{2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl}phenol (ZM241385), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS1706) were tested on wild-type and nine CAM A(2B) receptors with different levels of constitutive activity in a yeast growth assay. All three compounds turned out to be inverse agonists for the adenosine A(2B) receptor because they were able to fully reverse the basal activity of four low-level constitutively active A(2B) receptor mutants and to partially reverse the basal activity of three medium-level constitutively active A(2B) receptor mutants. We also discovered two highly constitutively active mutants whose basal activity could not be reversed by any of the three compounds. A two-state receptor model was used to explain the experimental observations; fitting these yielded the following relative intrinsic efficacies for the three inverse agonists ZM241385, DPCPX, and MRS1706: 0.14 +/- 0.03, 0.35 +/- 0.03, and 0.31 +/- 0.02, respectively. Moreover, varying L, the ratio of active versus inactive receptors in this model, from 0.11 for mutant F84L to 999 for two highly constitutively active mutants yielded simulated dose-response curves that mimicked the experimental curves. This study is the first description of inverse agonists for the human adenosine A(2B) receptor. Moreover, the use of receptor mutants with varying levels of constitutive activity enabled us to determine the relative intrinsic efficacy of these inverse agonists.

Adenosine A2 Receptor Agonists↗
PubMed1997

Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system.

The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

Animals↗
PubMed2000

Inverse agonism and constitutive activity as functional correlates of serotonin h5-HT(1B) receptor/G-protein stoichiometry.

This study evaluated the influence of receptor/G-protein (R:G) stoichiometry on constitutive activity and the efficacy of agonists, partial agonists, and inverse agonists at human (h) 5-hydroxytryphamine 1B (5-HT(1B)) receptors. Two Chinese hamster ovary cell lines were used; they expressed 8.5 versus 0.4 pmol h5-HT(1B) receptors/mg (determined by [(3)H]GR125,743 saturation analysis) and 3.0 versus 1.5 pmol receptor-activated G-proteins/mg [determined by guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) isotopic dilution], respectively. Thus, they displayed R:G ratios of approximately 3.0 (RGhigh) and approximately 0.3 (RGlow), respectively. In competition-binding experiments, the agonists, 5-HT and sumatriptan, displayed fewer high-affinity (HA)-binding sites and the partial agonists, BMS181, 101 and L775,606, displayed decreased affinity in RGhigh versus RGlow membranes. In contrast, the inverse agonists, SB224,289 and, to a lesser extent, methiothepin, showed increased affinity. In G-protein activation experiments, both basal and 5-HT-activated [(35)S]GTPgammaS binding were higher in RGhigh than in RGlow membranes. Constitutive activity (determined by inhibition of basal [(35)S]GTPgammaS binding with GTPgammaS in the absence of receptor ligands) was more pronounced in RGhigh versus RGlow membranes, as revealed by the >5-fold greater proportion of HA sites. Correspondingly, the negative efficacy of inverse agonists was strikingly augmented, inasmuch as they suppressed approximately two-thirds of HA [(35)S]GTPgammaS binding in RGhigh membranes, but only approximately one-third in RGlow membranes. Furthermore, the efficacy of partial agonists was greater at RGhigh versus RGlow membranes, as estimated by their ability to enhance [(35)S]GTPgammaS binding. In conclusion, an increase in R:G ratios at h5-HT(1B) receptors was associated with an increase in relative efficacy of partial agonists and, most notably, an increase in both constitutive G-protein activation and negative efficacy of inverse agonists.

Animals↗
PubMed2001

Inverse agonist up-regulates the constitutively active D3.49(164)Q mutant of the rat mu-opioid receptor by stabilizing the structure and blocking constitutive internalization and down-regulation.

We demonstrated previously that D3.49(164) mutations resulted in constitutive activation of the rat mu-opioid receptor and abolished receptor expression unless cells were pretreated with naloxone, an inverse agonist. In this study, we investigated the properties of the D3.49(164)Q mutant and the mechanisms underlying the effect of naloxone. Naloxone pretreatment up-regulated [(3)H]diprenorphine binding and protein expression of the D3.49(164)Q mutant in a time- and dose-dependent manner without affecting its mRNA level. After naloxone removal, binding and protein expression of the mutant declined with time with no effect on its mRNA level. Naloxone methiodide (a quaternary ammonium analog) caused a maximal up-regulation about 50% of the naloxone effect, indicating that naloxone acts extracellularly and intracellularly. Expression of the mutant was enhanced by inverse agonists, a neutral antagonist, and agonists, with inverse agonists being most effective. In membranes, the mutant was structurally less stable than the wild type upon incubation at 37 degrees C, and naloxone and [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin stabilized the mutant. Coexpression of the dominant-negative mutants GRK2-K220R, arrestin-2(319-418), dynamin I-K44A, rab5A-N133I or rab7-N125I partially prevented the decline in binding of the mutant after naloxone removal. Chloroquine or proteasome inhibitor I reduced the down-regulation of the mutant. These results indicate that the D3.49(164)Q mutant is constitutively internalized via G protein coupled-receptor kinase-, arrestin-2-, dynamin-, rab5-, and rab7-dependent pathways and probably trafficked through early and late endosomes into lysosomes and degraded by lysosomes and proteasomes. Naloxone up-regulates the D3.49(164)Q mutant by stabilizing the mutant protein and blocking its constitutive internalization and down-regulation. To the best of our knowledge, this represents the first comprehensive analysis of the mechanisms involved in up-regulation of constitutively active mutants by an inverse agonist.

Animals↗