Inflammation-associated changes in DOR expression and function in the mouse colon

Abstract

Endogenous opioids activate opioid receptors (ORs) in the enteric nervous system to control intestinal motility and secretion. The μ-OR mediates the deleterious side effects of opioid analgesics, including constipation, respiratory depression, and addiction. Although the δ-OR (DOR) is a promising target for analgesia, the function and regulation of DOR in the colon are poorly understood. This study provides evidence that endogenous opioids activate DOR in myenteric neurons that may regulate colonic motility. The DOR agonists DADLE, deltorphin II, and SNC80 inhibited electrically evoked contractions and induced neurogenic contractions in the mouse colon. Electrical, chemical, and mechanical stimulation of the colon evoked the release of endogenous opioids, which stimulated endocytosis of DOR in the soma and proximal neurites of myenteric neurons of transgenic mice expressing DOR fused to enhanced green fluorescent protein. In contrast, DOR was not internalized in nerve fibers within the circular muscle. Administration of dextran sulfate sodium induced acute colitis, which was accompanied by DOR endocytosis and an increased density of DOR-positive nerve fibers within the circular muscle. The potency with which SNC80 inhibited neurogenic contractions was significantly enhanced in the inflamed colon. This study demonstrates that DOR-expressing neurons in the mouse colon can be activated by exogenous and endogenous opioids. Activated DOR traffics to endosomes and inhibits neurogenic motility of the colon. DOR signaling is enhanced during intestinal inflammation. This study demonstrates functional expression of DOR by myenteric neurons and supports the therapeutic targeting of DOR in the enteric nervous system. NEW & NOTEWORTHY DOR is activated during physiologically relevant reflex stimulation. Agonist-evoked DOR endocytosis is spatially and temporally regulated. A significant proportion of DOR is internalized in myenteric neurons during inflammation. The relative proportion of all myenteric neurons that expressed DOR and the overlap with the nNOS-positive population are increased in inflammation. DOR-specific innervation of the circular muscle is increased in inflammation, and this is consistent with enhanced responsiveness to the DOR agonist SNC80.

Mu and Delta Opioid Receptors Are Coexpressed and Functionally Interact in the Enteric Nervous System of the Mouse Colon

“…The ability of MOR and DOR to functionally interact in the ENS and the potential mechanisms involved were determined by using receptor endocytosis assays and by pharmacologic examination of neurogenic contractions of the colon. 24 was detected in tissues from C57Bl/6J wild-type mice. MORmCherry labeling was most evident in myenteric neurons ileum and distal colon.…”

“…The activation of DOR by endogenous opioids is enhanced during both colonic inflammation and states of high intraluminal pressure. 24 This may affect the activity of endogenous MOR-acting ligands including enkephalins or endorphins.…”

“…Tissues for sectioning and whole mounts were prepared as described. 20,24 Target proteins were detected by indirect immunofluorescence using primary antibodies outlined in Table 3. Briefly, sections and whole mounts were incubated in blocking buffer (5% normal horse serum, 0.1% Triton X-100 in PBS containing 0.1% sodium azide; 1 hour, room temperature [RT]) and then labeled with primary antibodies (diluted in blocking buffer, 4 C, overnight).…”

“…Distal colons from C57Bl/6J mice were excised and prepared for tissue contraction assays of the circular muscle layer as described in detail. 24,27 Colons were placed in 10 mL water-jacketed organ baths containing Krebs solution (in mmol/L; NaCl 118, KCl 4.70, NaH 2 PO 4 .2H 2 O 1, NaHCO 3 25, MgCl 2 .6H 2 O 1.2, D-Glucose 11, CaCl 2 .2H 2 O 2.5) and maintained at 37 C and bubbled with 95% O 2 /5% CO 2 . Isometric contractions of the circular muscle were measured by a Grass FT03 force displacement transducer (Grass Instruments, Quincy, MA).…”

“…Neurogenic contractions were evoked by transmural electrical field stimulation (EFS) (0.5-msec duration, 3 pulses s -1 , 60 V), which was applied through platinum electrodes incorporated into the tissue holder. 24 Tissues were incubated with DMSO (0.1%), NLT (100 nmol/L), or CTOP (1 mmol/L) for the entire experiment. Once reproducible baseline responses were maintained (!3 sets, 5-minute intervals), tissues were treated cumulatively with agonists (1 nmol/L-10 mmol/L, 5 minutes).…”