Eva Martínez-Pinilla

Mechanisms of cannabidiol neuroprotection in hypoxic–ischemic newborn pigs: Role of 5HT1A and CB2 receptors

The mechanisms underlying the neuroprotective effects of cannabidiol (CBD) were studied in vivo using a hypoxic-ischemic (HI) brain injury model in newborn pigs. One- to two-day-old piglets were exposed to HI for 30 min by interrupting carotid blood flow and reducing the fraction of inspired oxygen to 10%. Thirty minutes after HI, the piglets were treated with vehicle (HV) or 1 mg/kg CBD, alone (HC) or in combination with 1 mg/kg of a CB₂ receptor antagonist (AM630) or a serotonin 5HT(1A) receptor antagonist (WAY100635). HI decreased the number of viable neurons and affected the amplitude-integrated EEG background activity as well as different prognostic proton-magnetic-resonance-spectroscopy (H(±)-MRS)-detectable biomarkers (lactate/N-acetylaspartate and N-acetylaspartate/choline ratios). HI brain damage was also associated with increases in excitotoxicity (increased glutamate/N-acetylaspartate ratio), oxidative stress (decreased glutathione/creatine ratio and increased protein carbonylation) and inflammation (increased brain IL-1 levels). CBD administration after HI prevented all these alterations, although this CBD-mediated neuroprotection was reversed by co-administration of either WAY100635 or AM630, suggesting the involvement of CB₂ and 5HT(1A) receptors. The involvement of CB₂ receptors was not dependent on a CBD-mediated increase in endocannabinoids. Finally, bioluminescence resonance energy transfer studies indicated that CB₂ and 5HT(1A) receptors may form heteromers in living HEK-293T cells. In conclusion, our findings demonstrate that CBD exerts robust neuroprotective effects in vivo in HI piglets, modulating excitotoxicity, oxidative stress and inflammation, and that both CB₂ and 5HT(1A) receptors are implicated in these effects.

Detection of cannabinoid receptors CB1 and CB2 within basal ganglia output neurons in macaques: changes following experimental parkinsonism

Although type 1 cannabinoid receptors (CB1Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R–CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R–CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R–CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers.

Health benefits of methylxanthines in cacao and chocolate.

One may wonder why methylxanthines are so abundant in beverages used by humans for centuries, or in cola-drinks that have been heavily consumed since their appearance. It is likely that humans have stuck to any brew containing compounds with psychoactive properties, resulting in a better daily life, i.e., more efficient thinking, exploring, hunting, etc., however, without the serious side effects of drugs of abuse. The physiological effects of methylxanthines have been known for a long time and they are mainly mediated by the so-called adenosine receptors. Caffeine and theobromine are the most abundant methylxanthines in cacao and their physiological effects are notable. Their health-promoting benefits are so remarkable that chocolate is explored as a functional food. The consequences of adenosine receptor blockade by natural compounds present in cacao/chocolate are here reviewed. Palatability and health benefits of methylxanthines, in general, and theobromine, in particular, have further contributed to sustain one of the most innocuous and pleasant habits: chocolate consumption.

l-DOPA disrupts adenosine A2A–cannabinoid CB1–dopamine D2 receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: Biochemical and behavioral studies

Long-term therapy with L-3,4-dihydroxyphenylalanine (L-DOPA), still the most effective treatment in Parkinsons disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating L-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1-D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A-CB1-D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A-CB1-D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by L-DOPA treatment. Radioligand binding data showed that A2A-CB1-D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of L-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in L-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with L-DOPA.

Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery.

l-DOPA-treatment in primates disrupts the expression of A2A adenosine–CB1 cannabinoid–D2 dopamine receptor heteromers in the caudate nucleus

The molecular basis of priming for L-DOPA-induced dyskinesias in Parkinsons disease (PD), which depends on the indirect pathway of motor control, is not known. In rodents, the indirect pathway contains striatopallidal GABAergic neurons that express heterotrimers composed of A(2A) adenosine, CB(1) cannabinoid and D(2) dopamine receptors that regulate dopaminergic neurotransmission. The present study was designed to investigate the expression of these heteromers in the striatum of a primate model of Parkinsons disease and to determine whether their expression and pharmacological properties are altered upon L-DOPA treatment. By using the recently developed in situ proximity ligation assay and by identification of a biochemical fingerprint, we discovered a regional distribution of A(2A)/CB(1) /D(2) receptor heteromers that predicts differential D(2)-mediated neurotransmission in the caudate-putamen of Macaca fascicularis. Whereas heteromers were abundant in the caudate nucleus of both naïve and MPTP-treated monkeys, L-DOPA treatment blunted the biochemical fingerprint and led to weak heteromer expression. These findings constitute the first evidence of altered receptor heteromer expression in pathological conditions and suggest that drugs targeting A(2A)-CB(1) -D(2) receptor heteromers may be successful to either normalize basal ganglia output or prevent L-DOPA-induced side effects.

CB1 and GPR55 receptors are co-expressed and form heteromers in rat and monkey striatum

Heteromerization of G-protein-coupled receptors is an important event as they integrate the actions of extracellular signals to give heteromer-selective ligand binding and signaling, opening new avenues in the development of potential drug targets in pharmacotherapy. The aim of the present paper was to check for cannabinoid CB1-GPR55 receptor heteromers in the central nervous system (CNS), specifically in striatum. First, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors, using bioluminescence resonance energy transfer and in situ proximity ligation assays (PLA). In the heterologous system, a biochemical fingerprint consisting on cross-antagonism in ERK1/2 phosphorylation was detected. The cross-antagonism was also observed on GPR55-mediated NFAT activation. Direct identification of GPR55 receptors in striatum is here demonstrated in rat brain slices using a specific agonist. Moreover, the heteromer fingerprint was identified in these rat slices by ERK1/2 phosphorylation assays whereas PLA assays showed results consistent with receptor-receptor interactions in both caudate and putamen nuclei of a non-human primate. The results indicate not only that GPR55 is expressed in striatum but also that CB1 and GPR55 receptors form heteromers in this specific CNS region.

GPR40 activation leads to CREB and ERK phosphorylation in primary cultures of neurons from the mouse CNS and in human neuroblastoma cells

GPR40, the free fatty acid receptor 1, is expressed strongly in the primate pancreas and brain. While the role of pancreatic GPR40 in glucose homeostasis has been extensively studied, the absence of this G‐protein‐coupled receptor from the brain of rodents has hampered studies into its role in the central nervous system. However, we found intense GPR40 mRNA expression by in situ hybridization in mouse hippocampal and motor cortex neurons. Furthermore, in a neuroblastoma cell GPR40 was activated by docosahexaenoic acid and selective agonists, yet not by palmitic acid. Significantly, the activation of GPR40 provoked the phosphorylation of the cAMP response element‐binding protein, CREB. The receptor was also functional in primary cultures of murine neurons, in which its activation by a selective agonist produced the phosphorylation of CREB and of extracellular signal‐regulated kinases, ERK1/2. These results suggest that mice represent a suitable model for elucidating the role of GPR40 in brain function.

Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum.

Identification of G protein-coupled receptors and their specific function in a given neuron becomes essential to better understand the variety of signal transduction mechanisms associated with neurotransmission. We hypothesized that angiotensin II type 1 (AT1) and dopamine D2 receptors form heteromers in the central nervous system, specifically in striatum. Using bioluminescence resonance energy transfer, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors. Heteromerization did not affect cAMP signaling via D2 receptors but attenuated the coupling of AT1 receptors to Gq. A common feature of heteromers, namely cross-antagonism, i.e. the blockade of the signaling of one receptor by the blockade of the partner receptor, was tested in co-transfected cells. Candesartan, the selective AT1 receptor antagonist, was able to block D2-receptor mediated effects on cAMP levels, MAP kinase activation and β-arrestin recruitment. This effect of candesartan, which constitutes a property for the dopamine-angiotensin receptor heteromer, was similarly occurring in primary cultures of neurons and rat striatal slices. The expression of heteromers in striatum was confirmed by robust labeling using in situ proximity ligation assays. The results indicate that AT1 receptors are expressed in striatum and form heteromers with dopamine D2 receptors that enable drugs selective for the AT1 receptor to alter the functional response of D2 receptors.

The relevance of theobromine for the beneficial effects of cocoa consumption

Cocoa consumption began in America and in the mid sixteenth Century it quickly spread to Europe. Beyond being considered a pleasant habit due to its rich sweet lingering taste, chocolate was considered a good nutrient and even a medicine. Traditionally, health benefits of cocoa have been related with the high content of antioxidants of Theobroma cocoa beans. However, the direct psychoactive effect due to methylxanthines in cocoa is notable. Theobromine and caffeine, in the proportions found in cocoa, are responsible for the liking of the food/beverage. These compounds influence in a positive way our moods and our state of alertness. Theobromine, which is found in higher amounts than caffeine, seems to be behind several effects attributed to cocoa intake. The main mechanisms of action are inhibition of phosphodiesterases and blockade of adenosine receptors. Further mechanisms are being explored to better understand the health benefits associated to theobromine consumption. Unlike what happens in other mammals -pets- included, theobromine is safe for humans and has fewer unwanted effects than caffeine. Therefore, theobromine deserves attention as one of the most attractive molecules in cocoa.