Physical and functional interaction between CB1 cannabinoid receptors and ��2-adrenoceptors

Background and purpose:

The CB₁ cannabinoid receptor and the β₂-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.

Experimental approach:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling.

Key results:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of β₂-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB₁ receptors. Co-expression altered the signalling properties of CB₁receptors, resulting in increased Gα_i-dependent ERK phosphorylation, but decreased non-Gα_i-mediated CREB phosphorylation. The CB₁ receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated β₂-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB₁ receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB₁ receptors and β₂-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the β₂-adrenoceptor–pERK response.

Conclusion and implications:

A complex interaction was demonstrated between CB₁ receptors and β₂-adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co-expressed.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Guidelines for reporting experiments involving animals: the ARRIVE guidelines

British Journal of Pharmacology (BJP) is pleased to publish a new set of guidelines for reporting research involving animals, simultaneously with several other journals; the ‘ARRIVE’ guidelines (Animals in Research: Reporting In Vivo Experiments). This editorial summarizes the background to the guidelines, gives our view of their significance, considers aspects of specific relevance to pharmacology, re-states BJP''s guidelines for authors on animal experiments and indicates our commitment to carrying on discussion of this important topic. We also invite feedback via the British Pharmacological Society website.     

Large normal and reduced penetrance alleles in Huntington disease: instability in families and frequency at the laboratory,at the clinic and in the population

Sequeiros J, Ramos EM, Cerqueira J, Costa MC, Sousa A, Pinto‐Basto J, Alonso I. Large normal and reduced penetrance alleles in Huntington disease: instability in families and frequency at the laboratory, at the clinic and in the population. Large normal (‘intermediate’) alleles may produce de novo expansions in Huntington disease; nevertheless, there is very little evidence about their population prevalence and impact in daily practice, and there are conflicting reports about the extent of their instability. We estimated the frequency of large normal alleles (27–35 CAGs) and of reduced penetrance alleles (36–39 CAGs), as well as the frequency of genotypes carrying them, in (i) a diagnostic laboratory, (ii) a genetic counselling clinic and (iii) the general population. Large normal alleles were present in 6% of a large control sample, 7% of consultands who took pre‐symptomatic testing and 7% of samples in the laboratory. Reduced penetrance alleles were found in 1 of 1772 control chromosomes (0.1% of individuals), 5% of 146 pre‐symptomatic testees and over 2% of 1214 diagnostic samples (350 families). All 16 alleles sized 27–32 CAGs seemed to be transmitted stably; alleles ≥ 36 repeats were unstable in five families. Seven small full penetrance alleles contracted into the reduced penetrance range, but none into the large normal range. Evidence showed that large normal alleles are relatively frequent and that those with reduced penetrance are not a rare event, either at the laboratory or the clinic. This reinforces the need to understand the genomic context of repeat instability in each family and population.     

Effect of low doses of cannabidiolic acid and ondansetron on LiCl-induced conditioned gaping (a model of nausea-induced behaviour) in rats

Background and Purpose

To determine the minimally effective dose of cannabidiolic acid (CBDA) that effectively reduces lithium chloride (LiCl)-induced conditioned gaping reactions (nausea-induced behaviour) in rats and to determine if these low systemic doses of CBDA (5–0.1 μg·kg⁻¹) relative to those of CBD could potentiate the anti-nausea effects of the classic 5-hydroxytryptamine 3 (5-HT₃) receptor antagonist, ondansetron (OND).

Experimental Approach

We investigated the efficacy of low doses of CBDA to suppress acute nausea, assessed by the establishment of conditioned gaping to a LiCl-paired flavour in rats. The potential of threshold and subthreshold doses of CBDA to enhance the reduction of nausea-induced conditioned gaping by OND were then determined.

Key Results

CBDA (at doses as low as 0.5 μg·kg⁻¹) suppressed nausea-induced conditioned gaping to a flavour. A low dose of OND (1.0 μg·kg⁻¹) alone reduced nausea-induced conditioned gaping, but when it was combined with a subthreshold dose of CBDA (0.1 μg·kg⁻¹) there was an enhancement in the suppression of LiCl-induced conditioned gaping.

Conclusions and Implications

CBDA potently reduced conditioned gaping in rats, even at low doses and enhanced the anti-nausea effect of a low dose of OND. These findings suggest that combining low doses of CBDA and OND will more effectively treat acute nausea in chemotherapy patients.     

Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases

Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases. To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [80%]) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a well- defined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.     

Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes

The localization of three known alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fg) has been studied in human megakaryocytes (MK) by immunofluorescence and immunoelectron microscopy. For this study, highly purified populations of MK were prepared from human bone marrow either by counterflow centrifugal elutriation or by cell culture from normal subjects and from two patients with megakaryoblastic leukemia. In normal bone marrow immature MK, TSP, and vWF were observed in the Golgi-associated vesicles and in small immature alpha-granules; in mature MK, they were found in the matrix of the mature large alpha-granules. Surprisingly, Fg was detected neither in the Golgi area, nor in the small precursors of alpha-granules; it was only found in the mature alpha-granules but this labeling was generally weaker than in blood platelets. In order to confirm these differences between the expression of Fg and vWF or TSP additional studies were performed on cultured maturing MK: immunofluorescent and ultrastructural immunogold labeling confirmed that vWF appeared early in the maturation while the same immature MK were negative for Fg. In the late maturation stage, the three proteins were detected in the alpha-granules. In order to know whether Fg was lately synthesized or endocytosed from the outside medium, normal MK were grown in the presence of either normal or afibrinogenemic plasma, and normal serum. Fg was detected only in the alpha-granules of MK grown in normal plasma. Similar results were observed with malignant MK, whose maturation was independent of the culture conditions. In conclusion, this study brings immunocytochemical evidence that vWF and TSP are synthesized by immature MK, whereas Fg appears later in the MK alpha-granules and its expression is dependent of the presence of an exogenous Fg source.     

Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF

Recombinant human interleukin-5 (rhIL-5), in either liquid or semi- solid cultures, selectively induced eosinophil production from normal human bone marrow, with no activity on other cell lineages. The time course of eosinophil production induced by murine IL-5, rhIL-3, and rh granulocyte-macrophage colony stimulating factor (GMCSF) was similar to rhIL-5. The rate of eosinophil maturation in vitro was independent of the stimulating cytokine, mature eosinophils being produced after 4 to 5 weeks in liquid culture with each of these cytokines. The eosinophils produced in response to each cytokine were morphologically indistinguishable, and had the ultrastructural features of maturity except that the electron-dense material in the granules had not formed into crystalline cores. Neither rhIL-1 nor rhIL-6 alone, or in combination with rhIL-5 or rhIL-3, induced eosinophil differentiation or proliferation under the conditions used. rhIL-3 and rhGMCSF induced more eosinophil colonies than rhIL-5, rhIL-5 had an additive, not synergistic, effect on eosinophil colony production when combined with either rhIL-3 or rhGMCSF, suggesting that rhIL-5 stimulates a smaller and possibly different population of eosinophil progenitors. However, rhIL-5 induced the greatest eosinophil production in liquid cultures, suggesting that although it may act on a smaller population of precursors, it is able to stimulate more proliferative steps than either rhIL-3 or rhGMCSF.