AB5636P Sigma-AldrichAnti-Cannabinoid Receptor 1 Antibody, NT

Brain mitochondrial activity is centrally involved in the central control of energy balance. When studying mitochondrial functions in the brain, however, discrepant results might be obtained, depending on the experimental approaches. For instance, immunostaining experiments and biochemical isolation of organelles expose investigators to risks of false positive and/or false negative results. As an example, the functional presence of cannabinoid type 1 (CB1) receptors on brain mitochondrial membranes (mtCB1) was recently reported and rapidly challenged, claiming that the original observation was likely due to artifact results. Here, we addressed this issue by directly comparing the procedures used in the two studies. Our results show that the use of appropriate controls and quantifications allows detecting mtCB1 receptor with CB1 receptor antibodies, and that, if mitochondrial fractions are enriched and purified, CB1 receptor agonists reliably decrease respiration in brain mitochondria. These data further underline the importance of adapted experimental procedures to study brain mitochondrial functions.

Cisplatin chemotherapy frequently causes severe vomiting in two temporally separated clusters of bouts dubbed the acute and delayed phases. Cannabinoids can inhibit the acute phase, albeit through a poorly understood mechanism. We examined the substrates of cannabinoid-mediated inhibition of both the emetic phases via immunolabeling for serotonin, Substance P, cannabinoid receptors 1 and 2 (CB(1), CB(2)), and the neuronal activation marker Fos in the least shrew (Cryptotis parva). Shrews were injected with cisplatin (10mg/kg i.p.), and one of vehicle, Delta(9)-THC, or both Delta(9)-THC and the CB(1) receptor antagonist SR141716A (2mg/kg i.p.), and monitored for vomiting. Delta(9)-THC-pretreatment caused concurrent decreases in the number of shrews expressing vomiting and Fos-immunoreactivity (Fos-IR), effects which were blocked by SR141716A-pretreatment. Acute phase vomiting induced Fos-IR in the solitary tract nucleus (NTS), dorsal motor nucleus of the vagus (DMNX), and area postrema (AP), whereas in the delayed phase Fos-IR was not induced in the AP at all, and was induced at lower levels in the other nuclei when compared to the acute phase. CB(1) receptor-IR in the NTS was dense, punctate labeling indicative of presynaptic elements, which surrounded Fos-expressing NTS neurons. CB(2) receptor-IR was not found in neuronal elements, but in vascular-appearing structures. All areas correlated with serotonin- and Substance P-IR. These results support published acute phase data in other species, and are the first describing Fos-IR following delayed phase emesis. The data suggest overlapping but separate mechanisms are invoked for each phase, which are sensitive to antiemetic effects of Delta(9)-THC mediated by CB(1) receptors.

To study the N-linked glycosylation properties of the CB1 receptor, rat brain membranes were treated with exo- and endoglycosidases. For visualizing CB1 receptors, an antipeptide antibody was raised against the N-terminal 14 amino acids and used to specifically detect the protein by Western blotting. We found that the apparent molecular weight of mature CB1 receptors was 64 kDa. Treatment of membranes with endoglycosidase F shifted the 64 kDa band to the 59 kDa and 53 kDa bands. The latter is consistent with the calculated molecular weight of deglycosylated CB1 receptors. Treatment of membranes with endoglycosidase H and alpha-mannosidase partially shifted the 64 kDa band to 53 kDa band, indicating a portion of the oligosaccharides was of the high mannose type. These data confirmed that the CB1 receptors in brain are N-linked glycoproteins with heterogeneous carbohydrate composition. Among three potential N-linked glycosylation sites on the N-terminus of the CB1 receptor, only two sites are actually glycosylated.