Receptor and G betagamma isoform-specific interactions with G protein-coupled receptor kinases.

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.

Information-theoretic analysis of a stimulated-Brillouin-scattering-based slow-light system.

We use an information-theoretic method developed by Neifeld and Lee [J. Opt. Soc. Am. A 25, C31 (2008)] to analyze the performance of a slow-light system. Slow-light is realized in this system via stimulated Brillouin scattering in a 2 km-long, room-temperature, highly nonlinear fiber pumped by a laser whose spectrum is tailored and broadened to 5 GHz. We compute the information throughput (IT), which quantifies the fraction of information transferred from the source to the receiver and the information delay (ID), which quantifies the delay of a data stream at which the information transfer is largest, for a range of experimental parameters. We also measure the eye-opening (EO) and signal-to-noise ratio (SNR) of the transmitted data stream and find that they scale in a similar fashion to the information-theoretic method. Our experimental findings are compared to a model of the slow-light system that accounts for all pertinent noise sources in the system as well as data-pulse distortion due to the filtering effect of the SBS process. The agreement between our observations and the predictions of our model is very good. Furthermore, we compare measurements of the IT for an optimal flattop gain profile and for a Gaussian-shaped gain profile. For a given pump-beam power, we find that the optimal profile gives a 36% larger ID and somewhat higher IT compared to the Gaussian profile. Specifically, the optimal (Gaussian) profile produces a fractional slow-light ID of 0.94 (0.69) and an IT of 0.86 (0.86) at a pump-beam power of 450 mW and a data rate of 2.5 Gbps. Thus, the optimal profile better utilizes the available pump-beam power, which is often a valuable resource in a system design.

High-fidelity, broadband stimulated-Brillouin-scattering-based slow light using fast noise modulation.

We demonstrate a 5-GHz-broadband tunable slow-light device based on stimulated Brillouin scattering in a standard highly-nonlinear optical fiber pumped by a noise-current-modulated laser beam. The noisemodulation waveform uses an optimized pseudo-random distribution of the laser drive voltage to obtain an optimal flat-topped gain profile, which minimizes the pulse distortion and maximizes pulse delay for a given pump power. In comparison with a previous slow-modulation method, eye-diagram and signal-to-noise ratio (SNR) analysis show that this broadband slow-light technique significantly increases the fidelity of a delayed data sequence, while maintaining the delay performance. A fractional delay of 0.81 with a SNR of 5.2 is achieved at the pump power of 350 mW using a 2-km-long highly nonlinear fiber with the fast noise-modulation method, demonstrating a 50% increase in eye-opening and a 36% increase in SNR in the comparison.

Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Collimation of a D-D Neutron Generator for Clinical Implementation of Neutron Stimulated Emission Computed Tomography: a Monte Carlo Study

This work is an investigation into collimator designs for a deuterium-deuterium (DD) neutron generator for an inexpensive and compact neutron imaging system that can be implemented in a hospital. The envisioned application is for a spectroscopic imaging technique called neutron stimulated emission computed tomography (NSECT).

Previous NSECT studies have been performed using a Van-de-Graaff accelerator at the Triangle Universities Nuclear Laboratory (TUNL) in Duke University. This facility has provided invaluable research into the development of NSECT. To transition the current imaging method into a clinically feasible system, there is a need for a high-intensity fast neutron source that can produce collimated beams. The DD neutron generator from Adelphi Technologies Inc. is being explored as a possible candidate to provide the uncollimated neutrons. This DD generator is a compact source that produces 2.5 MeV fast neutrons with intensities of 1012 n/s (4π). The neutron energy is sufficient to excite most isotopes of interest in the body with the exception of carbon and oxygen. However, a special collimator is needed to collimate the 4π neutron emission into a narrow beam. This work describes the development and evaluation of a series of collimator designs to collimate the DD generator for narrow beams suitable for NSECT imaging.

A neutron collimator made of high-density polyethylene (HDPE) and lead was modeled and simulated using the GEANT4 toolkit. The collimator was designed as a 52 x 52 x 52 cm3 HDPE block coupled with 1 cm lead shielding. Non-tapering (cylindrical) and tapering (conical) opening designs were modeled into the collimator to permit passage of neutrons. The shape, size, and geometry of the aperture were varied to assess the effects on the collimated neutron beam. Parameters varied were: inlet diameter (1-5 cm), outlet diameter (1-5 cm), aperture diameter (0.5-1.5 cm), and aperture placement (13-39 cm). For each combination of collimator parameters, the spatial and energy distributions of neutrons and gammas were tracked and analyzed to determine three performance parameters: neutron beam-width, primary neutron flux, and the output quality. To evaluate these parameters, the simulated neutron beams are then regenerated for a NSECT breast scan. Scan involved a realistic breast lesion implanted into an anthropomorphic female phantom.

This work indicates potential for collimating and shielding a DD neutron generator for use in a clinical NSECT system. The proposed collimator designs produced a well-collimated neutron beam that can be used for NSECT breast imaging. The aperture diameter showed a strong correlation to the beam-width, where the collimated neutron beam-width was about 10% larger than the physical aperture diameter. In addition, a collimator opening consisting of a tapering inlet and cylindrical outlet allowed greater neutron throughput when compared to a simple cylindrical opening. The tapering inlet design can allow additional neutron throughput when the neck is placed farther from the source. On the other hand, the tapering designs also decrease output quality (i.e. increase in stray neutrons outside the primary collimated beam). All collimators are cataloged in measures of beam-width, neutron flux, and output quality. For a particular NSECT application, an optimal choice should be based on the collimator specifications listed in this work.

Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans.

OBJECTIVE: The orexigenic gut hormone ghrelin and its receptor are present in pancreatic islets. Although ghrelin reduces insulin secretion in rodents, its effect on insulin secretion in humans has not been established. The goal of this study was to test the hypothesis that circulating ghrelin suppresses glucose-stimulated insulin secretion in healthy subjects. RESEARCH DESIGN AND METHODS: Ghrelin (0.3, 0.9 and 1.5 nmol/kg/h) or saline was infused for more than 65 min in 12 healthy patients (8 male/4 female) on 4 separate occasions in a counterbalanced fashion. An intravenous glucose tolerance test was performed during steady state plasma ghrelin levels. The acute insulin response to intravenous glucose (AIRg) was calculated from plasma insulin concentrations between 2 and 10 min after the glucose bolus. Intravenous glucose tolerance was measured as the glucose disappearance constant (Kg) from 10 to 30 min. RESULTS: The three ghrelin infusions raised plasma total ghrelin concentrations to 4-, 15-, and 23-fold above the fasting level, respectively. Ghrelin infusion did not alter fasting plasma insulin or glucose, but compared with saline, the 0.3, 0.9, and 1.5 nmol/kg/h doses decreased AIRg (2,152 +/- 448 vs. 1,478 +/- 2,889, 1,419 +/- 275, and 1,120 +/- 174 pmol/l) and Kg (0.3 and 1.5 nmol/kg/h doses only) significantly (P < 0.05 for all). Ghrelin infusion raised plasma growth hormone and serum cortisol concentrations significantly (P < 0.001 for both), but had no effect on glucagon, epinephrine, or norepinephrine levels (P = 0.44, 0.74, and 0.48, respectively). CONCLUSIONS: This is a robust proof-of-concept study showing that exogenous ghrelin reduces glucose-stimulated insulin secretion and glucose disappearance in healthy humans. Our findings raise the possibility that endogenous ghrelin has a role in physiologic insulin secretion, and that ghrelin antagonists could improve beta-cell function.

Acute administration of unacylated ghrelin has no effect on Basal or stimulated insulin secretion in healthy humans.

Unacylated ghrelin (UAG) is the predominant ghrelin isoform in the circulation. Despite its inability to activate the classical ghrelin receptor, preclinical studies suggest that UAG may promote β-cell function. We hypothesized that UAG would oppose the effects of acylated ghrelin (AG) on insulin secretion and glucose tolerance. AG (1 µg/kg/h), UAG (4 µg/kg/h), combined AG+UAG, or saline were infused to 17 healthy subjects (9 men and 8 women) on four occasions in randomized order. Ghrelin was infused for 30 min to achieve steady-state levels and continued through a 3-h intravenous glucose tolerance test. The acute insulin response to glucose (AIRg), insulin sensitivity index (SI), disposition index (DI), and intravenous glucose tolerance (kg) were compared for each subject during the four infusions. AG infusion raised fasting glucose levels but had no effect on fasting plasma insulin. Compared with the saline control, AG and AG+UAG both decreased AIRg, but UAG alone had no effect. SI did not differ among the treatments. AG, but not UAG, reduced DI and kg and increased plasma growth hormone. UAG did not alter growth hormone, cortisol, glucagon, or free fatty acid levels. UAG selectively decreased glucose and fructose consumption compared with the other treatments. In contrast to previous reports, acute administration of UAG does not have independent effects on glucose tolerance or β-cell function and neither augments nor antagonizes the effects of AG.