GTPγS Incorporation in the Rat Brain: A Study on μ-Opioid Receptors and CXCR4

Abstract

Chemokine and opioid receptors are G-protein-coupled receptors that play important roles in both the central nervous system and the immune system. The long-term goal of our research is to establish whether opioids regulate the activity of the chemokine receptor CXCR4 (one of the major HIV coreceptors) in the brain. In this research, we studied the anatomical distribution of functional receptors in young and adult animals by using the [ 35 S]GTPγS "binding" assay as an indication of G-protein activation by CXCL12 (the natural CXCR4 ligand) or by μ-opioid agonists. Brain slices or homogenates from Holtzmann rats of different ages (from 2 to 21 days old and adult animals) were treated with CXCL12 (0.001-100 nM), D-ala2,MePhe4,gly-ol5]enkephalin (DAMGO; 0.0003-10 μM) or morphine (0.0003-10 μM) and then processed for the assay. Our results show stimulation of both μ-OR and CXCR4 in several brain areas, including cortex and hippocampus (p<0.001); this effect is dose and age dependent, and the magnitude of response varies among different brain regions. Furthermore, AMD3100 (100 ng/ml), a specific CXCR4 antagonist, abolished CXCL12 stimulation in all the brain regions analyzed (p<0.001). Our findings suggest a similar pattern of expression for μ-OR and CXCR4 in the brain, supporting the possibility of an interaction between the two G-proteincoupled receptors in vivo. This might be relevant to the role of opiates in HIV neuropathogenesis.

Functions of the Chemokine Receptor CXCR4 in the Central Nervous System and Its Regulation by μ-Opioid Receptors

“…The μ-opioid receptor may also form heterodimers with CXCR4, but this has not been investigated as extensively as the δ-opioid receptor- CXCR4 heterodimer formation. In 3-week-old rats, guanosine 5’- O -[gamma-thio]triphosphate (GTPγS) studies show that μ-opioid receptors and CXCR4 are co-expressed in several different brain areas, including the medial and lateral cortex and the hippocampus (Burbassi, Aloyo, Simansky, & Meucci, 2008). Pretreatment with morphine or the μ-opioid receptor agonist DAMGO resulted in reduced GTPγS coupling after CXCL12 treatment (Burbassi et al, 2008).…”

“…In 3-week-old rats, guanosine 5’- O -[gamma-thio]triphosphate (GTPγS) studies show that μ-opioid receptors and CXCR4 are co-expressed in several different brain areas, including the medial and lateral cortex and the hippocampus (Burbassi, Aloyo, Simansky, & Meucci, 2008). Pretreatment with morphine or the μ-opioid receptor agonist DAMGO resulted in reduced GTPγS coupling after CXCL12 treatment (Burbassi et al, 2008). Interestingly, in μ-opioid receptor knockout glia, CXCL12-induced CXCR4 G protein coupling and other downstream signals are reduced, suggesting that regulatory mechanisms between these systems may differ depending on the cell type and the opioid receptor subtype (Burbassi, Sengupta, & Meucci, 2010).…”

“…This is associated with decreased activation of CXCR4 by CXCL12 and decreased activation of downstream signals, including ERK1/2 and Akt (Sengupta et al, 2009). Interestingly, FHC co-immunoprecipitates with CXCR4 after morphine treatment (Sengupta et al, 2009), which corresponds to reduced coupling of CXCR4 to G proteins (Burbassi et al, 2008). These findings suggest that FHC may interfere with G protein-mediated signals from CXCR4.…”

Contribution of SDF-1a/CXCR4 Signaling to Brain Development and Glioma Progression

“…SDF-1α can influence the pharmacodynamic action of neuronal active agents, such as the opioids and cannabinoids. A similar pattern of expression is found for CXCR4 and µ-, ĸ-, and δ-opioid receptors in the brain, including the cingulate cortex, hippocampus, and periaqueductal gray (PAG) [104,105,106,107]. As is known, the PAG is a brain region in charge of pain signal processes, a target which many analgesic compounds act on [108].…”

Interactions of HIV and Drugs of Abuse

“…Interestingly, although a subset of the patients sampled undoubtedly were substance abusers, many were not—suggesting that MOR receptor is inextricably linked to processes influencing HIV pathogenesis irrespective of opiate exposure. Although the mode of action is unclear, MOR activation can alter the expression of HIV-1 chemokine coreceptors involved in HIV entry, and MOR can undergo heterologous desensitization with CXCR4 (Szabo et al, 2002; Steele, Henderson, & Rogers, 2003; Patel et al, 2006; Finley et al, 2008; Burbassi, Aloyo, Simansky, & Meucci, 2008; Pitcher et al, 2014) or CCR5 (Chen et al, 2004; Happel, Steele, Finley, Kutzler, & Rogers, 2008; Song et al, 2011). Lastly, non-opioid genes may influence opiate drug and HIV interactions.…”