José Ángel Narváez

Intramembrane receptor-receptor interactions: a novel principle in molecular medicine

Summary.In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor–receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor–receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor–receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor–receptor interactions namely the presence of regulated cooperativity via receptor–receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor–receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor–receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor–receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson’s disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor–receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems.

Molecular phenotype of rat striatal neurons expressing the dopamine D5 receptor subtype

Dopamine is one of the principal neurotransmitters in the basal ganglia, where it plays a critical role in motor control and cognitive function through its interactions with the specific dopamine receptors D1 to D5. Although the activities mediated by most dopamine receptor subtypes have already been determined, the role of the D5 receptor subtype in the basal ganglia has still not been established. Furthermore, it is often difficult to distinguish between dopamine D5 and D1 receptors as they are stimulated by the same ligands, and they have a similar molecular structure and pharmacology. In an effort to understand the differences between these two receptor subtypes, we have studied the distribution of neurons containing D5 receptors in the striatum, and their molecular phenotype. As a result, we show that the D5 receptor subtype is present in two different populations of striatal neurons, projection neurons and interneurons. Overall, the abundance of this receptor subtype in the striatum is low, particularly in striatal projection neurons of both the direct and indirect projection pathways. In contrast, the expression of D5 receptors in striatal interneurons (cholinergic, somatostatin‐ or parvalbumin‐positive neurons) is high, while low to moderate expression was observed in calretinin‐positive neurons. Our results demonstrate the presence of D5 receptors in all the striatal cell populations so far described, although at different intensities in each. The fact that a large number of striatal neurons express the D5 receptor subtype suggests that this receptor fulfils an important function in the process of integrating information in the striatum.

Galanin receptor-1 modulates 5-hydroxtryptamine-1A signaling via heterodimerization.

Previous biochemical, cardiovascular and behavioral work has given evidence for the existence of antagonistic galanin receptor-5-HT1A receptor interactions in the brain. In this study we investigated the existence of GalR1-5-HT1A receptor heteromers and their functional characteristics. In mammalian cells transfected with GFP2-tagged 5-HT1A receptor and YFP-tagged GalR1 receptor, a proximity-based fluorescence resonance energy transfer technique was used and it has been demonstrated that GalR1-5-HT1A receptors heteromerize. Furthermore, signaling by either the mitogen-activated protein kinase (MAPK) or adenylyl cyclase (AC) pathways by these heteromers indicates a trans-inhibition phenomenon through their interacting interface via allosteric mechanisms that block the development of an excessive activation of G(i/o) and an exaggerated inhibition of AC or stimulation of MAPK activity. The presence of these heteromers in the discrete brain regions is postulated based on the existence of GalR-5-HT1A receptor-receptor interactions previously described in the brain and gives rise to explore possible novel therapeutic strategies for treatment of depression by targeting the GalR1-5-HT1A heteromers.

Extrasynaptic neurotransmission in the modulation of brain function. Focus on the striatal neuronal-glial networks.

Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks.

Neurotensin-induced modulation of dopamine D2 receptors and their function in rat striatum: counteraction by a NTR1-like receptor antagonist.

The present study investigated the neurotensin (NT) receptor subtype (NTR) involved in the antagonistic neurotensin modulation of striatal dopamine D2 receptors observed in vitro and in vivo. The NT induced increase of the IC50 values of dopamine (DA) competition for [125I]iodosulpiride binding sites was counteracted by the NTR1-like antagonist SR48692 in rat striatal slices. Intrastriatal perfusion of pergolide induced in the awake rat an inhibition of striatal DA release that was antagonized by NT. This action of NT was counteracted by co-perfusion with the NTR1 like antagonist SR48692. These data indicate that there exists in the striatum at the prejunctional level an intramembrane antagonistic NT receptor/DA D2 receptor-receptor interaction where NTR1 like receptor activation reduces the DA D2 autoreceptor function.

On the existence and function of galanin receptor heteromers in the central nervous system

Galanin receptor (GalR) subtypes 1–3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia–neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR–5-HT1A heteromers likely exist with antagonistic GalR–5-HT1A receptor–receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1–5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1–GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1–GalR2–5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR–5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR–NPYY1 receptor interactions in putative GalR–NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1–GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1–GalR2–NPYY2 heterotrimer. Finally, putative GalR–α2-adrenoreceptor heteromers with antagonistic receptor–receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression.

Centrally coinjected galanin and a 5-HT1A agonist act synergistically to produce vasodepressor responses in the rat

The present study was undertaken to evaluate the possible functional implications of the previously demonstrated in vitro interactions between galanin and 5-HT1A receptors. To this end we analysed the interactions between galanin and the 5-HT1A receptor agonist 8-OH-2-(di-n-propylamino)-tetralin (8-OH-DPAT) in central cardiovascular regulation. 8-OH-DPAT given intracisternally (i.c.) produced a dose-dependent reduction of blood pressure, the peak action being 32% at 10 nmol of 8-OH-DPAT. Heart rate and respiration rate were not affected. The vasodepressor action of 8-OH-DPAT was counteracted by the 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190). A threshold dose (1 nmol) of galanin given i.c. was shown to enhance the vasodepressor effect of both an ED50 dose and a threshold dose of 8-OH-DPAT. Quantitative receptor autoradiography showed that the IC50 values for [125I]galanin binding sites were reduced in the presence of 8-OH-DPAT (10 nM) by approximately 40% in the dorsal region of the nucleus of the solitary tract, the area postrema, and the raphe pallidus and obscurus nuclei. Galanin (10 nM) also significantly increased the IC50 value for [3H]8-OH-DPAT binding sites within the nucleus of the solitary tract. The results provide evidence for a synergistic interaction between 8-OH-DPAT and galanin in cardiovascular regulation after their central administration, an interaction possibly related to the ability of 8-OH-DPAT to enhance the affinity of the galanin receptor within regions of the medulla oblongata involved in cardiovascular control.

Role of galanin and galanin(1–15) on central cardiovascular control

Galanin and the N-terminal fragment Galanin(1-15) are involved in central cardiovascular regulation. The present paper reviews the recent cardiovascular results obtained by intracisternal injections of Galanin and Galanin(1-15) showing that: (A) the Galanin antagonist M40 blocks the central cardiovascular responses induced by Galanin(1-15) but not those elicited by Galanin; (B) both Galanin and Galanin(1-15) induce the expression of c-Fos in cardiovascular nuclei of the medulla oblongata with different temporal and spatial profiles; (C) the cardiovascular action of Galanin(1-15), but not Galanin, is mediated by peripheral beta-receptor stimulation; (D) and it is demonstrated an antagonistic Galanin/alpha2-adrenoceptors interaction as well as a differential modulation of central cardiovascular responses of Angiotensin II by Galanin or Galanin(1-15). All these data strengthen the involvement of both Galanin molecules as neuromodulators on central cardiovascular regulation.

Expression of D4 dopamine receptors in striatonigral and striatopallidal neurons in the rat striatum

Recent studies have reported the regional distribution of D(4) dopamine receptors in the rat striatum at the cellular and subcellular levels. However, the precise identity of the striatal neurons that express these receptors remains unknown. We have studied the expression of D(4) receptors in the striatal interneurons as well as in the output regions of the striatum using immunohistochemistry. Furthermore, we have evaluated the contribution of the striatum to D(4) receptor immunoreactivity in these areas by means of ibotenic acid lesion of the striatum. D(4) receptors were observed in the substantia nigra pars reticulata (SNr), the entopeduncular nucleus (EP) and the globus pallidus (GP), and they were found, using electron microscopy, to be located presynaptically. D(4) immunoreactivity in the striatal output nuclei was observed to dramatically decrease following lesion of the striatum with ibotenic acid. Striatal interneurons were not found to express D(4) receptors. These results demonstrate that D(4) receptors are located almost exclusively in striatal projection neurons, in both striatonigral and striatopallidal neurons.

Galanin/alpha2-receptor interactions in central cardiovascular control.

The modulation of the central cardiovascular effects of alpha(2)-adrenoceptor activation by galanin and its N-terminal fragment galanin-(1-15) has been evaluated by quantitative receptor autoradiography and cardiovascular analysis. Intracisternal coinjections of threshold doses of galanin and the selective and hypotensive alpha(2)-receptor agonist clonidine induced rapid and maintained vasopressor and tachycardic responses (p<0.001) instead of a hypotensive response, whereas the coinjections of threshold doses of the N-terminal galanin fragment (1-15) and clonidine did not elicit significant cardiovascular changes. Receptor autoradiographical experiments showed that galanin (1 nM) significantly increased the K(d) (p<0.01) and B(max) values (p<0.01) of [(3)H]p-Aminoclonidine binding sites in the nucleus tractus solitarii (NTS) compatible with a possible antagonistic interaction with the alpha(2)-adrenoceptors, and this effect was blocked by the presence of the specific galanin receptor antagonist M35. In addition, clonidine (30 nM) induced a 50% increase in the B(0) values of galanin based on competition experiments with [(125)I]-galanin binding in the NTS. These findings suggest the existence of an antagonistic effect of galanin, but not of galanin fragment (1-15), on the cardiovascular responses mediated by alpha(2)-receptors as well as a reciprocal facilitatory effect of alpha(2)-receptors on galanin binding. These mechanisms could be mediated by a reciprocal galanin-alpha(2) receptor interaction within the NTS.