Our pain marker-TRPV1-was recently referenced in an interesting study. Here researchers suggest that spatial distribution of the CB1 receptor and TRPV1 contributes to the complexity of their functional interaction.
They conclude: "Therefore, in order to utilise the analgesic potential of endogenous anandamide fully in PSN, ways to interfere with the mechanisms, through which the CB1 receptor promotes anandamide-responsiveness and sensitisation of TRPV1, must be identified. Due to the co-expression of TRPV1 and the CB1 receptor in various areas of the brain, the CB1 receptor – TRPV1 crosstalk we characterised in the present study could also have relevance in brain signal processing."
Chen J, Varga A, Selvarajah S, Jenes A, Dienes B, Sousa-Valente J, Kulik A, Veress G, Brain SD, Baker D, Urban L, Mackie K, Nagy I. Spatial Distribution of the Cannabinoid Type 1 and Capsaicin Receptors May Contribute to the Complexity of Their Crosstalk. Sci Rep. 2016 Sep 22;6:33307. doi: 10.1038/srep33307.
The SDS-FRL method reveals two major types of CB1 receptor and TRPV1 spatial distribution in DRG neurons.
(A,B) Double immunogold labelling for TRPV1 (12 nm particles; arrows) and the CB1 receptor (5 nm; arrowheads) reveals that the immunoreactivity for both proteins is moderate (A) to strong (B) on the protoplasmic face (P-face) but not on the exoplasmic face (E-face) of plasma membrane of PSN. In some patches of putative somatic membrane of PSN, the great majority of immunoparticles for TRPV1 and CB1 are isolated from each other (A), whereas on other patches of the membrane immunoparticles for TRPV1 and CB1 form co-clusters (arrow + arrowhead in B). Note the occasional co-clustering of particles labelling TRPV1 (double arrows in B). Scale bars = 0.2 μm
Methods: Sodium dodecyl sulfate (SDS)-digested freeze-fracture replica immunolabeling
Immunohisto-chemical labeling for electron microscopy was performed. Briefly, adult (150–200 g) male Sprague-Dawley rats (n = 4) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and perfused transcardially with 25 mM PBS, followed by 2% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB). Sections from DRGs were cut at a thickness of 110 μm. The slices were cryoprotected in a solution containing 30% glycerol in 0.1 M PB and then frozen by a high-pressure freezing machine (HPM 100; Leica, Austria). Frozen samples were inserted into a double replica table and fractured into two pieces at −130 °C and replicated by carbon deposition (5 nm thick), platinum (2 nm), and carbon (15 nm) in a freeze-fracture replica machine (BAF 060; BAL-TEC, Lichtenstein). They were digested in a solution containing 2.5% SDS and 20% sucrose in 15 mM Tris-HCl at 80 °C for 18 h then at 60 °C for 18 h. The replicas were washed in 25 mM PBS containing 0.05% bovine serum albumin (BSA; Roth, Germany) and then incubated in a blocking solution containing 5% BSA for 1 h. Subsequently, replicas were incubated in a mixture of primary antibodies (TRPV1 raised in guinea pig against the 22 amino acids of the C terminus (YTGSLKPEDAEVFKDSMVPGEK; Neuromics, USA), the dilution was 1:1000 and CB1 receptor (Prof. K. Mackie, raised in goat; Veress et al. 2014), dilution was 1:4000) in 50 mM Tris-buffered saline (TBS) containing 1% BSA. After several washes, replicas were reacted with a mixture of gold-coupled donkey anti-guinea pig (for TRPV1; 12 nm) and rabbit anti-goat (for CB1, 5 nm) secondary antibodies (1:30; BioCell Research Laboratories, Cardiff, UK) in 25 mM TBS containing 5% BSA. The anti-TRPV1 antibody from Neuromics provided better staining in the replicas than then the anti-TRPV1 antibody from Chemicon, which we tried in a pilot study. They were then washed and picked up on 100-mesh grids. The specificity of the antibodies was tested on tissues dissected from TRPV1 and CB1 knock out mice.
We will keep you posted as we discover new research on targeting CB1 for pain. That said, could this crosstalk be central to medical marijuana and its ability to ameliorate certain forms of pain?
We will keep you posted as we discover new research on targeting CB1 for pain. That said, could this crosstalk be central to medical marijuana and its ability to ameliorate certain forms of pain?
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