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PubMed1998

Three-dimensional spinal coupling mechanics: Part I. A review of the literature.

OBJECTIVE: To determine the state of knowledge relative to three-dimensional spinal coupled motion and to check the validity of currently accepted two-dimensional coupling as taught in chiropractic. DATA COLLECTION: A hand search of available reference texts and a computer search of literature from Index Medicus were collected with an emphasis on three-dimensional studies of human spinal movements. RESULTS: Most postural movements result in complicated three-dimensional spinal coupling in six degrees of freedom. Previous spinal coupling results based upon two-dimensional radiographic studies are inadequate and inaccurate. It is important that chiropractic colleges and techniques use the three-dimensional spinal kinematics to update their curricula and advance chiropractic treatment procedures. CONCLUSION: Full three-dimensional investigations of spinal coupling patterns have shown that the vertebrae rotate and translate in all three axes and that previous theories of spinal coupling based upon two-dimensional studies are inaccurate and invalid. Postural rotations and translations, which are the main motions studied in spinal coupling research, and altered configurations of the normal sagittal plane curves are the cause of both normal and abnormal spinal coupling patterns in three dimensions. Chiropractic letter listings (such as PRS, ASRP, etc.) are outdated, incomplete, invalid representations of coupled segmental movements. Mechanical loading of the neuromusculoskeletal tissues plays a vital role in position, dynamics, proper growth, repair and symptoms. Future studies of spinal kinematics should study the postural translations of the skull and thorax for their associated coupling in three dimensions. Combined postural rotations and translations along with altered sagittal curvatures need to be studied for their associated coupling characteristics as well.

Biomechanical Phenomena↗

PubMed1997

Condom and nonoxynol-9 use and the incidence of HIV infection in serodiscordant couples in Zambia.

We aimed to measure the effectiveness of latex condoms and of nonoxynol-9 [N-9] spermicides, in preventing HIV transmission in heterosexual serodiscordant couples in Lusaka. Each couple was examined at clinic visits scheduled at 3-month intervals for one year or more per couple, or until seroconversion or discontinuation. Couples were given condoms and their choice of 3 N-9 products and advised to use both at every intercourse. Sexual exposure was ascertained from coital logs that recorded coitus and barrier method use. HIV serological testing was done at each clinic visit (ELISA and Western blot if positive). One hundred and ten discordant couples were followed for a mean of 17.6 months. Seventy-eight per cent of coital episodes were protected by condoms, 85% by spermicides and 6.4% were unprotected. Fourteen seroconversions occurred (8.7 infections per 100 couple-years [c-y]). The rate was higher among seronegative men than seronegative women. Among couples who reported using condoms at every intercourse the infection rate was 2.3/100 c-y, compared with 10.7/100 c-y among couples using condoms less consistently (rate ratio [RR] 0.2; 95% confidence interval [CI] 0-1.6). Among couples who reported using N-9 at every intercourse, the seroconversion rate was 6.9/100 c-y; among couples who reported less than full-time N-9 use, the rate was 8.9/100 c-y (RR 0.8; 95% CI 0.2-2.8). Among the subset of female seronegatives, the N-9 RR was 0.5 (95% CI 0.1-3.8). But when we calculated HIV rates according to N-9 consistency in coital acts when condoms were not used, there was no evidence of protection with higher N-9 use. Consistent use of latex condoms reduces the incidence of HIV infection, but the association between N-9 spermicides and HIV is less clear. The current study could not provide compelling data on the impact of N-9 spermicide use on risk of HIV infection. The study's small size, as well as the consistency of concurrent condom use, limited our inferences. Available spermicide products must be studied further.

Adolescent↗

PubMed1999

Dark- and light-induced changes in coupling between horizontal cells in mammalian retina.

Retinal horizontal cells exhibit large receptive fields derived from their extensive electrical coupling by means of gap junctions. The conductance of these gap junctions seems to be regulated by dopamine acting through a cAMP-mediated cascade. There is now abundant evidence that extracellular dopamine levels vary with changes in ambient light intensity, suggesting that changes in the dark/light adaptational state of the retina can modulate coupling between horizontal cells. We studied this question in the mammalian retina by determining the effects of ambient light levels, in the form of changing background light intensity, on the coupling profiles of A- and B-type horizontal cells in the rabbit. Changes in coupling were assessed by measurements of the space constants of the syncytium formed by horizontal cells and the intercellular spread of the biotinylated tracer Neurobiotin. Our results indicate that dark-adapted horizontal cells show relatively weak coupling. However, presentation of background lights as dim as one-quarter log unit above rod threshold resulted in increases in both the averaged extent of tracer coupling and space constants of A- and B-type horizontal cells. Coupling expanded further as background light intensities were increased by 1-1.5 log units, after which additional light adaptation brought about an uncoupling of cells. Coupling reached its minimum at light intensities about 3 log units above rod threshold, after which, with further light adaptation, it stabilized at levels close to those seen in dark-adapted retinas. Our results indicate that electrical coupling between mammalian horizontal cells is modulated dramatically by changes in the adaptational state of the retina: coupling is maximized under dim ambient light conditions and diminishes as the retina is dark or light adapted from this level.

Adaptation, Ocular↗

PubMed1998

Effects of titanium-dental restorative alloy galvanic couples on cultured cells.

The potential exists for titanium and amalgams to become galvanically coupled in the oral cavity. While low galvanic corrosion rates have been measured in vivo for titanium-amalgam or mercury-free alloy couples, concerns exist over released corrosion products and adverse tissue responses. It was hypothesized in this study that coupling titanium to amalgams or gallium alloys increased the release of metallic corrosion products and decreased cellular activity and function. The effects of titanium coupled and uncoupled to a conventional amalgam, palladium-enriched spherical high copper amalgam, a dispersed type high copper amalgam, and a mercury-free gallium alloy were evaluated in 24-h cell culture tests. Viability, proliferation, and collagen synthesis were evaluated by the uptake of neutral red, 3H-thymidine, and immunoassay of procollagen, respectively, and compared to cells not exposed to any test material. The gallium alloy-titanium couple resulted in significant decreases in cellular viability, proliferation, and collagen synthesis as compared to the other coupled and uncoupled samples. Few differences in the cellular responses of the other coupled and uncoupled samples were observed. Atomic absorption analyses indicated increased release of metal ions from the amalgam and gallium alloy samples coupled to titanium as compared to their uncoupled condition, although the differences were not always significant. Galvanic corrosion of amalgam-titanium couples in the long term may become significant, and further research is needed. Coupling the gallium alloy to titanium may result in increased galvanic corrosion and cytotoxic responses.

Cells, Cultured↗

PubMed2000

Spin-spin coupling tensors in fluoromethanes

All spin-spin coupling tensors J of the fluoromethanes CH3F, CH2F2, and CHF3 are obtained theoretically by multiconfiguration self-consistent field linear response (MCSCF LR) ab initio calculations. Furthermore the principal values and the orientation of the principal axis systems of each theoretical J tensor are specified. Experimental liquid crystal NMR (LC NMR) data on the tensorial properties of the CF spin spin coupling in CH3F and CH2F2, and the FF spin-spin coupling in CHF3 are also reported. In the analysis of the experiments, the contributions from molecular vibrations, as well as that of the correlation of vibrational and rotational motion to the experimental anisotropic couplings, D(exp), are taken into account. The information of the anisotropic indirect coupling, 1/2J(aniso), is detected as the difference between D(exp) and the calculated dipolar coupling, D(calc). The extracted indirect contributions, 1/2J(aniso), are in fair agreement with the ab initio results. All relative (experimental and theoretical) CF and FF indirect contributions, 1/2J(aniso)/D(exp), are negative and under 1.7% in magnitude, when the observed molecular orientations are used. Therefore, in the one bond CF couplings and in the two bond FF couplings, the indirect contribution can normally be ignored without introducing serious error to the determination of molecular orientation and/or structure. However, a more accurate method is to partially correct for the indirect contribution by utilising the transferability of the spin-spin coupling tensors in related molecules. This is due to the fact that even small contributions may be significant, if the order parameter of the internuclear direction is negligibly small, leading to dominating indirect contributions. The very good agreement of the experimental values with the calculated coupling constants and the reasonable agreement in the anisotropic properties, which are experimentally much more difficult to define, indicates that the MCSCF LR method is capable of producing reliable J tensors for these systems, contrary to the case of density-functional theory.

Journal Article↗

PubMed2000

Rhythmic coupling among cells in the suprachiasmatic nucleus.

In mammals, the part of the nervous system responsible for most circadian behavior can be localized to a pair of structures in the hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies suggest that the basic mechanism responsible for the generation of these rhythms is intrinsic to individual cells. There is also evidence that the cells within the SCN are coupled to one another and that this coupling is important for the normal functioning of the circadian system. One mechanism that mediates coordinated electrical activity is direct electrical connections between cells formed by gap junctions. In the present study, we used a brain slice preparation to show that developing SCN cells are dye coupled. Dye coupling was observed in both the ventrolateral and dorsomedial subdivisions of the SCN and was blocked by application of a gap junction inhibitor, halothane. Dye coupling in the SCN appears to be regulated by activity-dependent mechanisms as both tetrodotoxin and the GABA(A) agonist muscimol inhibited the extent of coupling. Furthermore, acute hyperpolarization of the membrane potential of the original biocytin-filled neuron decreased the extent of coupling. SCN cells were extensively dye coupled during the day when the cells exhibit synchronous neural activity but were minimally dye coupled during the night when the cells are electrically silent. Immunocytochemical analysis provides evidence that a gap-junction-forming protein, connexin32, is expressed in the SCN of postnatal animals. Together the results are consistent with a model in which gap junctions provide a means to couple SCN neurons on a circadian basis.

Animals↗

PubMed1993

Morphological and electrophysiological evidence for electrotonic coupling of rat dorsolateral septal nucleus neurons in vitro.

Intracellular injections of Lucifer Yellow were utilized to evaluate the incidence of dye-coupling among dorsolateral septal nucleus (DLSN) neurons recorded from slice preparations of adult rat septal nuclei. Twenty percent of single injections of Lucifer Yellow resulted in pairs of labeled neurons. These dye-coupled cells were morphologically heterogeneous and did not exhibit any morphological characteristics that could be used to distinguish them from non dye-coupled neurons. The spatial separation of cell bodies and close apposition of dendrites within each pair indicated that the dye transfer site(s) were situated at dendrodendritic and/or dendrosomatic rather than somatosomatic junctions. The main axon of some dye-coupled neurons gave rise to intrinsic axon collaterals prior to exiting the nucleus indicating that these coupled neurons function as projection neurons as well as local circuit interneurons. Electrophysiological recordings of the passive membrane properties and spontaneous activity of individual dye-coupled neurons revealed no significant difference from non dye-coupled cells in the DLSN. Some neurons exhibited spontaneously occurring fast potentials which presumably represent electrotonic potentials. These fast potentials were often tightly coupled with action potentials but could be distinguished from synaptic potentials by their shape and their lack of voltage-dependent changes in amplitude. These morphological and supportive electrophysiological data provide the first indirect evidence for electrotonic coupling of dorsolateral septal neurons. The functional significance of this coupling may lie in the potential for synchronization of the output of the DLSN which could play an important role in the septal maintenance and modulation of hippocampal Theta rhythm.

Animals↗

PubMed1997

Interaction of Silane Coupling Agents with CaCO3

A study of eight silane coupling agents showed very different effect of these compounds on the mechanical properties of PP/CaCO3 composites. The application of aminofunctional silane coupling agents resulted in the reactive coupling of the two inactive components leading to increased strength and decreased deformability. A detailed study of the interaction between CaCO3 and the various coupling agents was carried out in order to find an explanation for the strong coupling effect. The amount of coupling agent creating a monolayer coverage was determined by a dissolution method for each coupling agent. The obtained values changed between 0.3 and 1.0 wt% calculated for the CaCO3 . An attempt was made to determine the orientation of the adsorbed molecules to the filler surface. Most of the coupling agents are oriented perpendicularly to the surface with the exception of a methacryl functional silane compound. Possible interactions between hydrolyzed or condensed silane coupling agents and the filler were studied by Fourier transform infrared spectroscopy using transmitting (FTIR-TS) and diffuse reflectance (DRIFT) modes, as well as gel permeation chromatography (GPC). The results showed that bulky organofunctional groups form a caged, polycyclic, low-molecular-weight structure on the surface, while silanes with smaller groups tend to condense into open, ladder type, high-molecular-weight polysiloxane chains. Polymer/filler adhesion, however, depends primarily on the chemical character of the organofunctional group. Aminofunctional silane coupling agents adhere well to the filler surface and react also with the polymer. In the case of similar functionality the size of the organofunctional group determines the strength of the adhesion.

Journal Article↗

PubMed2002

Measurement of small one-bond proton-carbon residual dipolar coupling constants in partially oriented (13)C natural abundance oligosaccharide samples: analysis of heteronuclear (1)J(CH)-modulated spectra with the BIRD inversion pulse.

Two 2D J-modulated HSQC-based experiments were designed for precise determination of small residual dipolar one-bond carbon-proton coupling constants in (13)C natural abundance carbohydrates. Crucial to the precision of a few hundredths of Hz achieved by these methods was the use of long modulation intervals and BIRD pulses, which acted as semiselective inversion pulses. The BIRD pulses eliminated effective evolution of all but (1)J(CH) couplings, resulting in signal modulation that can be described by simple modulation functions. A thorough analysis of such modulation functions for a typical four-spin carbohydrate spin system was performed for both experiments. The results showed that the evolution of the (1)H-(1)H and long-range (1)H-(13)C couplings during the BIRD pulses did not necessitate the introduction of more complicated modulation functions. The effects of pulse imperfections were also inspected. While weakly coupled spin systems can be analyzed by simple fitting of cross peak intensities, in strongly coupled spin systems the evolution of the density matrix needs to be considered in order to analyse data accurately. However, if strong coupling effects are modest the errors in coupling constants determined by the "weak coupling" analysis are of similar magnitudes in oriented and isotropic samples and are partially cancelled during dipolar coupling calculation. Simple criteria have been established as to when the strong coupling treatment needs to be invoked.

Carbon↗

PubMed1995

Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures.

Several coupling methods for binding antibodies (Ab) to liposomes have previously been developed. We were interested in examining if some of these methods would be suitable for attaching Ab to long-circulating formulations of liposomes (SL), sterically stabilized with poly(ethylene glycol) (PEG). We studied three 'classical' coupling methods in which Ab was attached at the bilayer surface of SL, and two new coupling methods in which Ab was attached at the PEG terminus. Parameters examined including binding efficiency, antibody surface density, the ability of the immunoliposomes to remote-load the anticancer drug doxorubicin, and the specific binding of the resulting immunoliposomes to target cells. The non-covalent biotin-avidin coupling method resulted in low Ab densities at the cell surface, as did a coupling in method in which maleimide-derivatized Ab was attached to the liposome surface through a thiolated phospholipid incorporated into the liposomes. The low levels of Ab achieved in these method was likely due to interference by PEG with the access of the Ab to the liposome surface. However, when a maleimide-derivatized Ab was coupled to thiolated PEG, moving the coupling reaction away from the liposome surface, very high coupling efficiencies were achieved, and these immunoliposomes achieved good specific binding to their target cells. Oxidizing the Fc region of the Ab and coupling it to the PEG terminus through a hydrazone bond was a less efficient coupling method, but had the advantage of retaining Ab orientation. Efficient remote-loading of doxorubicin was found for immunoliposomes in which Ab was attached at the PEG terminus.

Adenocarcinoma↗

PubMed1993

Electrotonic coupling between neurosecretory cells in the crayfish eyestalk.

Electrical coupling is described among neurosecretory cells in the crayfish X organ. By simultaneous impalements from pairs of neurons, only 30% were found to be coupled. All of them showed non-rectifying junctions. In all cases, electrical coupling corresponded to dye-coupling, as explored with intracellular injection of Lucifer yellow to one of the recorded cells. Some neurons were coupled to more than one neighbour. From dye fills it was apparent that the coupling site could be located far away from the cell body, immediately before the axonal branchings in the neuropile. The input resistance of the coupled neurons was consistently lower (24 +/- 16 M omega) than that of the non-coupled neurons (58 +/- 18 M omega). Synchronous synaptic activity was commonly recorded from coupled neurons, thus suggesting a functional significance of coupling in the integration of neurosecretory activity.

Action Potentials↗

PubMed1994

Incidence of neuronal coupling in supraoptic nuclei of virgin and lactating rats: estimation by neurobiotin and lucifer yellow.

Dye coupling among neurons has been shown to reflect electrotonic coupling. Recent work in retina has revealed that the incidence of coupling is greater when estimated by neurobiotin (NB) transfer than by Lucifer yellow (LY). Several previous studies have shown that the incidence of LY coupling among supraoptic nucleus (SON) neurons of lactating rats is 2- to 4-fold higher than is observed in virgin females. We compared the incidence of coupling among SON neurons following simultaneous injections of LY and NB into the same cells in slices from virgin or lactating rats. As seen in previous studies, there were 4-fold more LY-coupled neurons per injection in lactating as compared to virgin rats. Under both conditions, the numbers of NB-coupled neurons per injection were 4-fold greater than was observed for LY; possible mechanisms are discussed. Individual NB-filled neurons were coupled to as many as 10 other cells distributed over a large area of the SON. These results confirm previous findings of more coupling in lactating than virgin SONs, and suggest that both the incidence and spatial extent of interneuronal coupling are greater and thus probably more important functionally than has been heretofore appreciated.

Animals↗

PubMed1982

Coupling in rat hippocampal slices: dye transfer between CA1 pyramidal cells.

Intracellular injections of Lucifer Yellow-CH (LY) into CA1 pyramidal cells were made in rat hippocampal slices to study dye transfer between neurons as evidence that these cells are electrotonically coupled. Extensive control procedures were performed which substantially reduced inadvertent staining. Over half of the neurons were dye-coupled after injections in stratus pyramidale. Dye coupling occurred even when spike amplitudes were greater than or equal to 70 mV throughout the impalement and was still present after chemical synapses were blocked with a low Ca2+ solution containing Mn2+. Somata of dye-coupled cells were usually located within 35 micrometers (post-fixation) of the injected cell and showed no preferred orientation. Fast prepotentials and dye coupling occurred independently. Neurons in superior cervical ganglia, which were sliced and injected using similar procedures, showed no dye coupling. Intradendritic injections of LY in stratum radiatum also yielded dye coupling between CA 1 pyramidal cells, although the dye coupling was less frequent. Within stratum radiatum, neither extracellular ejections nor intracellular injections of interneurons were associated with multiple staining. Thus, injection of LY into the soma or dendrite of a single CA1 pyramidal cell often resulted in multiple staining, and in many ensembles the somata were well spaced. Control experiments suggested that such dye transfer is not by an extracellular route. This implied that some CA1 cells are electrotonically coupled. Further electrophysiological and morphological studies are required to resolve the discrepancies among various techniques used to evaluate the amount of coupling in the hippocampus.

Animals↗

PubMed2005

Effect of foot orthotics on rearfoot and tibia joint coupling patterns and variability.

The purpose of this study was to compare joint coupling patterns and variability of the rearfoot and tibia during running in subjects who were treated with two types of orthotic devices to that of controls. Eleven subjects with various lower extremity injuries were treated unsuccessfully with a standard orthotic, and then successfully with an inverted orthotic. Three-dimensional kinematic data were collected while subjects ran without orthoses and then in standard and inverted orthoses. Eleven healthy subjects ran without orthoses for comparison. The rearfoot inversion/eversion and tibial internal/external rotation joint coupling pattern and variability relationship was assessed using a vector coding technique. It was hypothesized that when the treated runners ran without orthotic devices, they would exhibit lower joint coupling angles and lower joint coupling variability compared to the controls. In addition, it was hypothesized that there would be no difference in the coupling angle or coupling variability between the standard and no orthotic conditions of the treated runners. Finally, it was hypothesized that coupling angle would decrease and variability would increase in the inverted versus the standard and non-orthotic conditions. No significant differences in joint coupling pattern or variability were observed between the treated and control subjects. In addition, no significant differences were noted between the orthotic conditions in the treated group. These results suggest that foot orthotic devices do not produce significant changes in rearfoot-tibial coupling. Therefore, the relief experienced with the inverted orthotic is likely due to factors other than alterations in this coupling.

Adolescent↗

PubMed2004

Structural characterization of unfolded states of apomyoglobin using residual dipolar couplings.

The conformational propensities of unfolded states of apomyoglobin have been investigated by measurement of residual dipolar couplings between (15)N and (1)H in backbone amide groups. Weak alignment of apomyoglobin in acid and urea-unfolded states was induced with both stretched and compressed polyacrylamide gels. In 8 M urea solution at pH 2.3, conditions under which apomyoglobin contains no detectable secondary or tertiary structure, significant residual dipolar couplings of uniform sign were observed for all residues. At pH 2.3 in the absence of urea, a change in the magnitude and/or sign of the residual dipolar couplings occurs in local regions of the polypeptide where there is a high propensity for helical secondary structure. These results are interpreted on the basis of the statistical properties of the unfolded polypeptide chain, viewed as a polymer of statistical segments. For a folded protein, the magnitude and sign of the residual dipolar couplings depend on the orientation of each bond vector relative to the alignment tensor of the entire molecule, which reorients as a single entity. For unfolded proteins, there is no global alignment tensor; instead, residual dipolar couplings are attributed to alignment of the statistical segments or of transient elements of secondary structure. For apomyoglobin in 8 M urea, the backbone is highly extended, with phi and psi dihedral angles favoring the beta or P(II) regions. Each statistical segment has a highly anisotropic shape, with the N-H bond vectors approximately perpendicular to the long axis, and becomes weakly aligned in the anisotropic environment of the strained acrylamide gels. Local regions of enhanced flexibility or chain compaction are characterized by a decrease in the magnitude of the residual dipolar couplings. The formation of a small population of helical structure in the acid-denatured state of apomyoglobin leads to a change in sign of the residual dipolar couplings in local regions of the polypeptide; the population of helix estimated from the residual dipolar couplings is in excellent agreement with that determined from chemical shifts. The alignment model described here for apomyoglobin can also explain the pattern of residual dipolar couplings reported previously for denatured states of staphylococcal nuclease and other proteins. In conjunction with other NMR experiments, residual dipolar couplings can provide valuable insights into the dynamic conformational propensities of unfolded and partly folded states of proteins and thereby help to chart the upper reaches of the folding landscape.

Acrylic Resins↗

PubMed2004

J-modulated ADEQUATE (JM-ADEQUATE) experiment for accurate measurement of carbon-carbon coupling constants.

A new method for the accurate determination of carbon-carbon coupling constants is described. The method is based on a modified ADEQUATE experiment, where a J-modulated spin-echo sequence precedes the ADEQUATE pulse scheme. The J-modulation and scaling of carbon-carbon couplings is based on simultaneous incrementation of 13C chemical shift and coupling evolution periods. The time increment for the homonuclear carbon-carbon coupling evolution can be suitably scaled with respect to the corresponding increment for the chemical shift evolution. Typically a scaling factor of 2 to 3 is employed for the measurement of one-bond coupling constants, while multiplication by a factor of 10 to 15 is applied when small long-range couplings are determined. The same pulse scheme with coupling evolution period optimized for one-bond or long-range couplings allows the determination of the corresponding carbon-carbon coupling constants. The splittings of the ADEQUATE crosspeaks in the F1 dimension yield the appropriately multiplied coupling constants.

Algorithms↗

PubMed2004

Synaptic integration in electrically coupled neurons.

Interactions among chemical and electrical synapses regulate the patterns of electrical activity of vertebrate and invertebrate neurons. In this investigation we studied how electrical coupling influences the integration of excitatory postsynaptic potentials (EPSPs). Pairs of Retzius neurons of the leech are coupled by a nonrectifying electrical synapse by which chemically induced synaptic currents flow from one neuron to the other. Results from electrophysiology and modeling suggest that chemical synaptic inputs are located on the coupled neurites, at 7.5 microm from the electrical synapses. We also showed that the space constant of the coupled neurites was 100 microm, approximately twice their length, allowing the efficient spread of synaptic currents all along both coupled neurites. Based on this cytoarchitecture, our main finding was that the degree of electrical coupling modulates the amplitude of EPSPs in the driving neurite by regulating the leak of synaptic current to the coupled neurite, so that the amplitude of EPSPs in the driving neurite was proportional to the value of the coupling resistance. In contrast, synaptic currents arriving at the coupled neurite through the electrical synapse produced EPSPs of constant amplitude. This was because the coupling resistance value had inverse effects on the amount of current arriving and on the impedance of the neurite. We propose that by modulating the amplitude of EPSPs, electrical synapses could regulate the firing frequency of neurons.

Action Potentials↗

PubMed1997

G protein coupling of the rat A1-adenosine receptor--partial purification of a protein which stabilizes the receptor-G protein association.

A membrane protein identified in cortical brain membranes and termed 'coupling cofactor', modulates G protein-coupling of the A1-adenosine receptor by reducing the catalytic efficiency of the receptor. Coupling cofactor traps the A1-adenosine receptor in the high affinity complex and, thus, is responsible for the resistance of high affinity A1-agonist binding to modulation by guanine nucleotides. In the present work, this effect was used for assaying the activity of coupling cofactor by reconstituting guanine-nucleotide resistant agonist binding to rat A1-adenosine receptors in detergent extracted brain membranes or in membranes from 293 cells after stable transfection with receptor cDNA. Coupling cofactor was partially purified from porcine brain membranes. The specific activity was modestly enriched (approximately 5-fold) after three chromatographic steps (DEAE-Sephacel, AcA34, MonoQ pH 8). Rechromatography of coupling cofactor over MonoQ at pH 7 resulted in a loss in specific activity if membranes of 293 cells but not if brain membranes were used as acceptor membranes. In addition, the molecular mass estimated by gel filtration decreased from > 150 kDa in the initial stage of purification to 40-30 kDa after this fourth chromatographic step. These two observations suggest that coupling cofactor requires an additional component that is present in brain membranes and is lost in later stages of purification. The activity of partially purified preparations of coupling cofactor activity relied also on the abundance of G protein alpha-subunits in the membrane. The activity on reconstitution with brain membranes or pertussis toxin pretreated 293 membranes was supported by addition of Gi alpha (rank order of protency: alpha i1 > alpha i3 > alpha i2) but not of G(o alpha). The selectivity for G protein alpha-subunits suggests that coupling cofactor may provide for an additional level of specificity in organizing receptor-G protein coupling.

Animals↗