Age-related loss of GPR68-expressing neurons within the mucosa, not the myenteric plexus of human colon

Objective: The G protein-coupled receptor 68 (GPR68) detects variations in extracellular pH, finding potential roles in homeo- stasis and responses to ischemia and inflammation within dif- ferent organs, including the gastrointestinal tract.[1] In human colon the distribution of GPR68 is unclear. We examined the lo- calization and density of GPR68 within the enteric nervous sys- tem of ascending (AC) and descending (DC) human colon from younger and older adults. Methods: Macroscopically normal AC and DC were ob- tained from patients undergoing lower bowel cancer resection (aged 22–91years; grouped into younger (≤60years) and older (≥ 67 years) populations). Immunolabelling was performed using formalin-fixed, paraffin-embedded sections and antibodies against GPR68, protein gene product 9.5 (PGP9.5) and calretinin to identify the presence and density of GPR68-immunoreactive (IR) expressing cells. Analysis used ImageJ. Results: Ageing did not change the density of total PGP9.5-IR enteric neuronal fibres in the AC or DC. For the myenteric plexus (MP) of both age groups, the densities of calretinin-IR neurons were similar in both the AC (younger adult: 1.2 ±0.3 × 10−3; older: 0.9 ± 0.2 × 10−3 per mm2 plexus) and DC (1.4 ± 0.2 × 10−3; 1.3 ± 0.3 × 10−3 per mm2 plexus), but reduced in the mu- cosa of older adults for both AC (respectively, 9.8 ±0.5 vs. 3.2 ±0.1/pixel) and DC (11.5 ±09 vs. 7.4 ±0.3/pixel). GPR68 was widely expressed in PGP9.5-positive nerve fibers across the mu- cosa, muscle and myenteric plexus of both the AC and DC. The density of GPR68-IR in the muscle and myenteric plexus was similar in both age groups but smaller in the mucosa of older adults for both AC and DC. AC (Younger adult, n=5; Older n=7) and DC (Younger, n=8; Older n=5). Mean±SEM. NS: non-significant. *P≤0.05. student's independent t-test 126 of 171 Conclusions: GPR68 is widely distributed within the enteric nervous system of the human colon, with potential roles for GPR68 suggested in the muscle and MP, and in the functions of calretinin-IR neurons within the mucosa. Further, the concom- itant loss of GPR68 and calretinin-IR neurons in the mucosa of older adults suggests selective vulnerability of mucosal sensory and homeostatic mechanisms of the ageing colon. 1Bajestani et al. Eur J Pharmacol 2024;978:176762