Search PubMed⌕ Search

Biomedical subjects

Kjell Fuxe

Publications and source records attributed to Kjell Fuxe.

At least 37 records · Page 2Linked to original sources
PubMed2005

Protection but maintained dysfunction of nigral dopaminergic nerve cell bodies and striatal dopaminergic terminals in MPTP-lesioned mice after acute treatment with the mGluR5 antagonist MPEP.

The mGluR5 antagonist MPEP was used to study the role of mGluR5 in MPTP-induced injury of the nigrostriatal DA neurons. The findings indicate that acute blockade of mGluR5 may result in neuroprotective actions against MPTP neurotoxicity on nigral DA cell bodies and striatal DA terminals using stereological analysis of TH immunoreactivity and microdensitometry. Biochemical analysis showed no restoration of DA levels and metabolism indicating a maintained reduction of DA transmission.

Animals↗
PubMed2005

How receptor mosaics decode transmitter signals. Possible relevance of cooperativity.

It has been demonstrated that receptor-receptor interactions between G-protein-coupled receptors (GPCRs) occur at the plasma-membrane level. It has also been shown that clustering of GPCRs in aggregates or receptor mosaics (RMs) results in the reciprocal modulation of their binding and decoding characteristics. It is hypothesized that cooperativity plays an important part in the decoding of signals processed by RMs of GPCRs. Thus, the binding of the ligand at one receptor alters the likelihood of the same ligand binding at the next site, in the case of RMs, formed by identical receptors and/or by iso-receptors (receptors that bind the same ligand).

Allosteric Regulation↗
PubMed2005

Dimer-based model for heptaspanning membrane receptors.

The existence of intramembrane receptor-receptor interactions for heptaspanning membrane receptors is now fully accepted, but a model considering dimers as the basic unit that binds to two ligand molecules is lacking. Here, we propose a two-state-dimer model in which the ligand-induced conformational changes from one component of the dimer are communicated to the other. Our model predicts cooperativity in binding, which is relevant because the other current models fail to address this phenomenon satisfactorily. Our two-state-dimer model also predicts the variety of responses elicited by full or partial agonists, neutral antagonists and inverse agonists. This model can aid our understanding of the operation of heptaspanning receptors and receptor channels, and, potentially, be important for improving the treatment of cardiovascular, neurological and neuropsychyatric diseases.

Animals↗
PubMed2005

Molecular mechanisms involved in the adenosine A and A receptor-induced neuronal differentiation in neuroblastoma cells and striatal primary cultures.

Adenosine A1 receptors (A1Rs) and adenosine A(2A) receptors (A(2A)Rs) are the major mediators of the neuromodulatory actions of adenosine in the brain. In the striatum A1Rs and A(2A)Rs are mainly co-localized in the GABAergic striatopallidal neurons. In this paper we show that agonist-induced stimulation of A1Rs and A(2A)Rs induces neurite outgrowth processes in the human neuroblastoma cell line SH-SY5Y and also in primary cultures of striatal neuronal precursor cells. The kinetics of adenosine-mediated neuritogenesis was faster than that triggered by retinoic acid. The triggering of the expression of TrkB neurotrophin receptor and the increase of cell number in the G1 phase by the activation of adenosine receptors suggest that adenosine may participate in early steps of neuronal differentiation. Furthermore, protein kinase C (PKC) and extracellular regulated kinase-1/2 (ERK-1/2) are involved in the A1R- and A(2A)R-mediated effects. Inhibition of protein kinase A (PKA) activity results in a total inhibition of neurite outgrowth induced by A(2A)R agonists but not by A1R agonists. PKA activation is therefore necessary for A(2A)R-mediated neuritogenesis. Co-stimulation does not lead to synergistic effects thus indicating that the neuritogenic effects of adenosine are mediated by either A1 or A(2A) receptors depending upon the concentration of the nucleoside. These results are relevant to understand the mechanisms by which adenosine receptors modulate neuronal differentiation and open new perspectives for considering the use of adenosine agonists as therapeutic agents in diseases requiring neuronal repair.

Adenosine↗
PubMed2005

New methods to evaluate colocalization of fluorophores in immunocytochemical preparations as exemplified by a study on A2A and D2 receptors in Chinese hamster ovary cells.

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein-coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor-receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

Adenosine A2 Receptor Agonists↗
PubMed2005

Trafficking of adenosine A2A and dopamine D2 receptors.

An interaction between adenosine A2A and dopamine D2 receptors has been demonstrated previously. It is generally found that agonist treatment internalizes receptors, including A2A and D2, whereas less is known of the long-term effects involved in the agonist-mediated trafficking of A2A and D2 receptors. Furthermore, the possible influence of the antagonists on receptor trafficking is still undefined. The present studies focus on the long-term effects of A2A and D2 agonist and D2 antagonist treatments on both A2A and D2 receptor trafficking studied at three different time intervals--3, 15, and 24 h. In addition, with the fluorescence resonance energy transfer technique, formation of heteromeric A2A and D2 receptor complexes was shown in the cotransfected CHO cell line. Confocal microscopy analysis showed that a 3-h treatment with the D2 agonist induced coaggregation of A2A/D2 receptors. These A2A/D2 receptor coaggregates internalized after 15 h with a recruitment of the receptors back to the cell membrane after 24 h. In contrast to the effects of the agonist treatment, a 3-h treatment with the D2-like antagonist raclopride increased both A2A and D2 immunoreactivity, indicating that the D2 antagonist stabilizes the D2 receptor and thereby reduces the internalization of both of the A2A and D2 receptors. Taken together, an activation of either A2A and D2 receptor or blockade of D2 receptors will cause long-lasting changes in A2A and D2 receptor trafficking.

Adenosine↗
PubMed2005

Role of electrostatic interaction in receptor-receptor heteromerization.

Using pull-down and mass spectrometry experiments, we have previously demonstrated that adenosine A2A-dopamine D2 receptor-receptor heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the third intracellular loop of the D2 receptor (217RRRRKR222) and two adjacent Asp residues (DD401-402) or a phosphorylated Ser (pS374) residue in the carboxyl terminus of the A2A receptor. It has been demonstrated recently that a specific region in the carboxyl terminus of the dopamine D1 receptor (L387-L416) and a specific region in the carboxyl terminus of the NR1-1 subunit of the NMDA receptor (E834-S938) are involved in D1-NMDA receptor-receptor heteromerization. Careful perusal of these interacting regions shows the presence of a phosphorylated serine (pS397) and adjacent glutamates (EE404-405) in the D1 receptor, whereas NR1-1 contains three adjacent Arg residues (RRR893-896). These epitopes are highly conserved in all species, a sign that the epitopes are likely to be involved in a physiologically significant activity. If similar epitopes are found to be involved in the formation of receptor heteromers other than A2A-D2 and D1-NMDA, the epitope-epitope electrostatic interaction might represent an important general mechanism underlying receptor- receptor interactions.

Amino Acid Sequence↗
PubMed2005

How proteins come together in the plasma membrane and function in macromolecular assemblies: focus on receptor mosaics.

Some theoretical aspects on structure and function of proteins have been discussed previously. Proteins form multimeric complexes, as they have the capability of binding other proteins (Lego property) resulting in multimeric complexes capable of emergent functions. Multimeric proteins might have either a genomic or a postgenomic origin. Proteins spanning the plasma membrane have been analyzed by considering the effects of the microenvironment in which the protein is embedded. In particular, the different effects of the hydrophilic (extracellular and intracellular) versus the lipophilic (intramembrane) environment have been considered. These aspects have been discussed in the framework of membrane microdomains, in particular, the so-called rafts. In alpha-helix proteins the individual peptide dipoles align to produce a macrodipole crossing the entire membrane. This macrodipole has its positive (extracellular) pole at the N-terminal end of the helix and its negative (intracellular) pole at the C-terminal end. This arrangement has been analyzed in the framework of the counter-ion atmosphere, that is, the formation of a cloud of small ions bearing an opposite charge. Excitable cells reverse their resting potential during the all-or-none action potentials. Hence, the extracellular side of the plasma membrane becomes negative with respect to the intracellular side. This change of polarization affects also the direction and magnitude of the alpha-helix dipole in view of the fact that there is a displacement of the counter ions. The oscillation in the intensity of the dipole caused by the action potentials opens the possibility of an interaction among dipoles by electromagnetic waves.

Animals↗
PubMed2005

Computer-assisted image analysis of caveolin-1 involvement in the internalization process of adenosine A2A-dopamine D2 receptor heterodimers.

A functional aspect of horizontal molecular networks has been investigated experimentally, namely the heteromerization between adenosine A2A and dopamine D2 receptors and the possible role of caveolin-1 in the co-trafficking of these molecular complexes. This study has been carried out by means of computer-assisted image analysis procedure of laser images of membrane immunoreactivity of caveolin-1, A2A, D1, and D2 receptors obtained in two clones of Chinese hamster ovary cells one transfected with A2A and dopamine D1 receptors and the other one with A2A and D2 receptors. Cells were treated for 3 h with 10 microM D1 receptor agonist SKF 38393, 50 microM D2-D3 receptor agonist quinpirole, and 200 nM A2A receptor agonist CGS 21680. In A2A-D1-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D1 receptors and treatment with SKF 38393 induced internalization of caveolin-1 and D1 receptors, with a preferential internalization of D1 receptors colocalized with caveolin-1. In A2A-D2-cotransfected cells, caveolin-1 was found to colocalize with both A2A and D2 receptors and either CGS 21680 or quinpirole treatment induced internalization of caveolin-1 and A2A and D2 receptors, with a preferential internalization of A2A and D2 receptors colocalized with caveolin-1. The results suggest that A2A and D2 receptors and caveolin-1 likely interact forming a macrocomplex that internalizes upon agonist treatment. These observations are discussed in the frame of receptor oligomerization and of the possible functional role of caveolin-1 in the process of co-internalization and, hence, in controlling the permanence of receptors at the plasma membrane level (prerequisite for receptor mosaic organization and plastic adjustments) and in the control of receptor desensitization.

Animals↗
PubMed2005

Receptor-receptor interactions, receptor mosaics, and basic principles of molecular network organization: possible implications for drug development.

The phenomenon of receptor-receptor interactions was hypothesized by Agnati and Fuxe in the 1980s, and several indirect proofs were provided in the following years by means of in vitro binding experiments and in vivo experiments in physiological and pathological animal models. This paper aims to outline some of the most important features and consequences of this phenomenon in the frame of the structural and functional aspects of molecular networks. In particular, the concepts of receptor mosaic (RM), and of horizontal and vertical molecular networks (HMNs, VMNs, respectively) are illustrated. To discuss some aspects of the functional organization of molecular networks, not only new data on protein-protein interactions but also the biochemical mechanism of cooperativity will be used. On this basis, some theoretical deductions can be drawn that allow a tentative classification of the RMs and the proposal of the extension of the concept of branching point introduced for enzymes to the possible switching role of some RMs in directing signals to various VMNs. Finally, the cooperativity phenomenon and the so-called symmetry rule will be used to introduce a proper mathematical approach that characterizes RMs as to their receptor composition, receptor topography, and order of receptor activation inside the RM. These new data on G protein-coupled receptors and molecular network organization indicate possible new approaches for drug development.

Animals↗
PubMed2005

Adenosine A2A and dopamine D2 heteromeric receptor complexes and their function.

The existence of A2A-D2 heteromeric complexes is based on coimmunoprecipitation studies and on fluorescence resonance energy transfer and bioluminescence resonance energy transfer analyses. It has now become possible to show that A2A and D2 receptors also coimmunoprecipitate in striatal tissue, giving evidence for the existence of A2A-D2 heteromeric receptor complexes also in rat striatal tissue. The analysis gives evidence that these heteromers are constitutive, as they are observed in the absence of A2A and D2 agonists. The A2A-D2 heteromers could either be A2A-D2 heterodimers and/or higher-order A2A -D2 hetero-oligomers. In striatal neurons there are probably A2A-D2 heteromeric complexes, together with A2A-D2 homomeric complexes in the neuronal surface membrane. Their stoichiometry in various microdomains will have a major role in determining A2A and D2 signaling in the striatopallidal GABA neurons. Through the use of D2/D1 chimeras, evidence has been obtained that the fifth transmembrane (TM) domain and/or the I3 of the D2 receptor are part of the A2A-D2 receptor interface, where electrostatic epitope-epitope interactions involving the N-terminal part of I3 of the D2 receptor (arginine-rich epitope) play a major role, interacting with the carboxyl terminus of the A2A receptor. Computerized modeling of A2A-D2 heteromers are in line with these findings. It seems likely that A2A receptor-induced reduction of D2 receptor recognition, G protein coupling, and signaling, as well as the existence of A2A-D2 co-trafficking, are the consequence of the existence of an A2A-D2 receptor heteromer. The relevance of A2A-D2 heteromeric receptor complexes for Parkinson's disease and schizophrenia is emphasized as well as for the treatment of these diseases. Finally, recent evidence for the existence of antagonistic A2A-D3 heteromeric receptor complexes in cotransfected cell lines has been summarized.

Animals↗
PubMed2005

Partners for adenosine A1 receptors.

G protein-coupled receptors (GPCRs) are targets for therapy in a variety of neurological diseases. Using adenosine A1 receptors (A1Rs) as paradigm of GPCRs, this review focuses on how protein-protein interactions, from monomers to heteromers, can contribute to hormone/neurotransmitter/neuromodulator regulation. The interaction of A1Rs with other membrane receptors, enzymes, and adaptor and scaffolding proteins is relevant for receptor traffic, internalization, and desensitization, and A1Rs are extremely important in driving signaling through different intracellular pathways. There is even the possibility of linking together GPCR heteromeric complexes with ion channel receptors in a receptor mosaic that might have special integrative value and might constitute the molecular basis for learning and memory.

Adenosine Deaminase↗
PubMed2005

Concluding remarks.

This meeting has well illustrated the state-of-the-art phenomenon of receptor-receptor interactions between heptaspanning membrane receptors (also named G protein-coupled receptors [GPCRs]), as both theoretical aspects and new experimental evidence have been discussed extensively.From a general standpoint the theoretical discussion has enlightened us to the phenomenon of receptor-receptor interactions as a "privileged window," which allows the exploration of some important structural and functional aspects of molecular networks (Agnati et al., this issue). Also, unexpected functions of well-known proteins can be studied, such as beta-arrestins, which are at the interface between horizontal and vertical molecular networks and have a signaling function of their own besides modulating receptor transduction and trafficking (Lefkowitz, this issue). The experimental data have shown that many in vitro approaches are now available to study the structure and function of homo- and hetero-oligomers in cell lines and primary cultures, making possible a major expansion of the field. However, the in vivo approaches to study the role of heteromeric receptor complexes in cell function have been less clear cut. It was discussed, especially by George, that one problem with receptor knockout mice is inter alia that the removal of one receptor will alter the balance of various homomeric and heteromeric receptor complexes in which the deleted receptor participated. In fact, each receptor might participate in a number of heteromeric receptor complexes. An attractive in vivo approach in the receptor-receptor interaction study will be to use knockin mice in which one receptor A (Ra) has been removed and replaced by a mutated A receptor (Ra'). Several different strategies can be considered, and here are some of them. 1. Ra' can still recognize its ligand and signal but does not interact with its usual heteromeric receptor partner B (Rb). Thus, Ra' operates as a monomer and/or homomer. 2. Ra' can still recognize its ligand and interact with Rb, but it cannot signal (e.g., it does not couple to the G protein). Thus, Ra' operates as a modulator protein of Rb. 3. Ra' can still recognize its ligand and signal, but it allosterically affects its heteromeric partner Rb in a different way. Thus, Ra' operates as an unusual modulator of Rb.

Animals↗
PubMed2005

Existence and theoretical aspects of homomeric and heteromeric dopamine receptor complexes and their relevance for neurological diseases.

Dopamine (DA) and other receptors physically interact in the plasma membrane of basal ganglia neurons forming receptor mosaics (RMs). Two types of RMs are discussed, homomers formed only by DA-receptor (DA-R) subtypes and heteromers formed by DA-R associated with other receptors, such as A2A, A1, mGluR5, N-methyl-D-aspartate (NMDA), gamma-aminobutryic acid (GABA)-A, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. By being part of horizontal molecular networks, RMs tune multiple effector systems already at membrane level, such as G protein regulated inward rectifying potassium channels and dopamine transporter activity. Also, ligand-gated ion channels such as GABA-A and NMDA receptors are modulated by DA-R, e.g., in the striatal GABA output neurons through the formation of heteromeric complexes with these receptors. Thus, intramembrane DA-R-receptor interactions play an important role in the information handling in the basal ganglia. On this basis, functional implications of DA RM in physiological and pathological conditions are discussed. The effects of temperature on RM are discussed not only because receptor-decoding mechanisms are temperature sensitive, but also in view of the suggestion that possible ordering effects (i.e., changes in the entropy of a receptor complex) induced by a ligand are as a result of alterations in the receptor oligomerization (i.e., are related to rearrangements of the RM). Hence, brain temperature may have profound effects on brain integrative functions not only because its effects on the kinetics of biochemical reactions, but also for its effects on receptor geometry, building up of RM, and alterations in protein expression, as is the case of H-channels following febrile seizures.

Animals↗
PubMed2004

Kinetic and functional properties of [3H]ZM241385, a high affinity antagonist for adenosine A2A receptors.

We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.

Adenosine↗
PubMed2004

Anxiolytic effects of intra-amygdaloid injection of the D1 antagonist SCH23390 in the rat.

The intercalated islands are intra-amigdaloid clusters of D1 receptor rich GABAergic neurons, which control impulse traffic between the basolateral complex and the central nucleus of the amygdala. As dopaminergic transmission within the amygdala may play a role in anxiety, the effect of the D1 antagonist SCH23390 microinjected mainly close to the rostral intercalated islands in rats was studied, using the White and Black Box test. SCH23390 reduced anxiety by an increase in the latency of the first entry into the black compartment and by an increase in the total time spent in the white compartment of the White and Black Box test, while there was no significant modification of locomotion. It is suggested that blockade of D1 receptors in the rostral intercalated islands may reduce anxiety through a reduction of GABA-mediated dishinibition of the central amygdaloid nucleus.

Amygdala↗
PubMed2004

Adenosine A2A receptor and dopamine D3 receptor interactions: evidence of functional A2A/D3 heteromeric complexes.

Adenosine A(2A) and dopamine D(2) receptors have been shown previously to form heteromeric complexes and interact at the level of agonist binding, G protein coupling, and trafficking. Because dopamine D(2) and D(3) receptors show a high degree of sequence homology, A(2A) and D(3) receptors may also interact in a similar manner. The present studies with confocal microscopy showed that A(2A)-yellow fluorescent protein (YFP) and D(3)-green fluorescent protein 2 (GFP2) receptors colocalize in the plasma membrane. Furthermore, fluorescence resonance energy transfer (FRET) analysis demonstrated that A(2A)-YFP and D(3)-GFP2 receptors give a positive FRET efficiency and are thereby likely to exist as heteromeric A(2A)/D(3) receptor complexes. Saturation experiments with [(3)H]dopamine demonstrated that the A(2A) receptor agonist 4-[2-[[6-amino-9(N-ethyl-beta-d-ribofuranuronaminoamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS-21680) reduced the affinity of the high-affinity agonist binding state of the D(3) receptor for [(3)H]dopamine. The A(2A) and D(2A) receptors seem to interact also at the level of G protein coupling, because the adenosine A(2A) receptor agonist CGS-21680 fully counteracted the D(3) receptor-mediated inhibition of a forskolin-mediated increase in cAMP levels. Taken together, when coexpressed in the same neuron, A(2A) and D(3) receptors seem to form A(2A)/D(3) heteromeric receptor complexes in which A(2A) receptors antagonistically modulate both the affinity and the signaling of the D(3) receptors. D(3) receptor is one of the therapeutic targets for treatment of schizophrenia, and therefore, the A(2A)/D(3) receptor interactions could provide an alternative antischizophrenic treatment.

Adenosine↗