Introduction

Inflammatory bowel disease (IBD) is characterised by inflammatory flares with excruciating pain, for which effective analgesics are lacking. Cocaine- and amphetamine-regulated transcript (CART) peptide regulates peripheral pain in mice.¹ However, its role in visceral nociception as well as the underlying molecular signalling mechanism in primary sensory neurons remains elusive. CART is highly expressed in TRPV1-positive colon-projecting sensory neurons.² In addition, the enzyme prohormone convertase 1/3, which is involved in processing CART into its bioactive fragment 55-102, is upregulated in colonic biopsies of IBD patients (internal data), suggesting a potential role for the peptide in colonic pain.

Aims & Methods

Here, we aimed to test if CART would affect firing of the colon-innervating lumbar splanchnic nerve (LSN) as model of visceral nociception and characterise its effect and signalling pathway in sensory neurons innervating the colon. Studies were performed using tissue from male and female C57BL/6J mice euthanised by cervical dislocation and exsanguination in accordance with Schedule 1 of the Animals (Scientific Procedures) Act 1986. Thoracolumbar dorsal root ganglia (DRG) neurons were dissociated and cultured overnight for Ca²⁺ imaging using Fluo-4 AM as previously described.³ Cells were perfused with extracellular solution (ECS) alone or supplemented with either rat CART 55-102, (20-300nM, ECS), thapsigargin (10mM, DMSO), xestospongin C (1mM, ECS), edelfosine (10mM, ECS), capsaicin (10mM, ethanol) or KCl solution (60mM, ddH₂O) for respective challenge or pre-treatment. All conditions were blinded and randomised. Colorectum with attached lumbar splanchnic nerve was carefully removed and mounted on a tissue bath superfused with Krebs buffer as previously described.³ Rat CART 55-102 was superfused at 5ml/min, resulting in a final concentration of 200nM. Statistical analysis was performed using Student´s t test or ANOVA and Dunnett´s multiple comparisons test. Data is displayed as mean ± SEM.

Results

CART elicited a concentration-dependent increase in intracellular Ca²⁺ in DRG neurons, which at the highest concentration tested (300nM) induced a significant increase in intracellular Ca²⁺ in 29.2 ± 2.98% of DRG neurons relative to 8.35 ± 1.25% of vehicle responders (p < 0.0001). 55.05% of CART-responsive neurons were putative nociceptors due to co-sensitivity to capsaicin. Further investigation revealed a statistically significant reduction in the proportion of CART-sensitive neurons compared to CART (300nM) alone following pre-treatment with edelfosine, a phospholipase C inhibitor (10.5 ± 3.88%, p = 0.006), thapsigargin, a SERCA pump inhibitor (7.25 ± 1.78%, p = 0.001), or xestospongin C, an inositol triphosphate receptor inhibitor (6.74± 1.87%, p = 0.006). Furthermore, tissue bath application of CART (200nM) elicited a marked transient increase in ongoing LSN discharge, which increased from a baseline of 12.8 ± 3.9 (spikes/s) to a peak firing of 19.7 ± 6.1 spikes/s (p = 0.0385).

Conclusion

CART stimulates intracellular Ca²⁺ release in capsaicin-sensitive neurons consistent with a role in pain processing. This response was mediated by the release of Ca²⁺ from intracellular stores such as the endoplasmic reticulum and attenuated by inhibitors of the G_q pathway, suggesting activation of a G_q-coupled GPCR. The pronociceptive activity of CART on nerve firing suggests a potential role in colonic pain. Further research is necessary to identify the role of CART in IBD.

References

1. Ohsawa M, Dun SL, Tseng LF, Chang J, Dun NJ. Decrease of hindpaw withdrawal latency by cocaine- and amphetamine-regulated transcript peptide to the mouse spinal cord. Eur J Pharmacol. Jul 7 2000;399(2-3):165-9. doi:10.1016/s0014-2999(00)00374-5
2. McGuire C, Boundouki G, Hockley JRF, et al. Ex vivo study of human visceral nociceptors. Gut. Jan 2018;67(1):86-96. doi:10.1136/gutjnl-2016-311629
3. Higham JP, Bhebhe CN, Gupta RA, et al. Transcriptomic profiling reveals a pronociceptive role for angiotensin II in inflammatory bowel disease. Pain. Jan 29 2024;doi:10.1097/j.pain.0000000000003159

Disclosure

The PhD research is partially funded by Metrion Biosciences.