Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons

Abstract

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

Cultured Vagal Afferent Neurons as Sensors for Intestinal Effector Molecules

“…Previous studies of VANs involving patch-clamp or calcium imaging interrogation were primarily performed in acute settings (less than 24 h in vitro [ 15 , 22 , 24 , 26 , 31 , 32 ]). Extracellular recordings, on the other hand, require longer culture durations for the cells to attach to or near the electrodes.…”

“…In addition to the surface coatings, two media types were also compared. A common media composition used for acute cultures of VANs is DMEM supplemented with 10% FBS (referred to as serum-based media) [ 15 , 22 , 24 , 26 ]. We hypothesized that a more specialized neuronal culture medium would better sustain neuronal cell viability and function in the non-acute cultures.…”

“…Others have shown that VANs respond to several effector molecules, such as gut hormones and paracrine mediators. For example, cholecystokinin (CCK), a peptide hormone produced from the gastrointestinal tract in response to the digestion of fat and protein, depolarizes VANs both in vivo and in vitro [ 21 , 22 ]. Serotonin (5-HT), an important neurotransmitter, also depolarizes VANs [ 23 ].…”

Vagal interoception of microbial metabolites from the small intestinal lumen

“…This may align with prior studies demonstrating that circulating BAs mediate release of cholecystokinin (CCK), and that CCK signaling is dynamic and rapidly desensitizes. 66 In contrast, perfusion of SCFAs into the SI lumen increased afferent vagal nerve activity following a latency period in an FFAR2-dependent manner ( Figure 3D-F ), suggesting indirect activation of FFAR2-expressing epithelial cells and subsequent GLP-1 release. 67 Finally, we found that microbiome-dependent 3IS in the small intestine elicited sustained afferent vagal nerve activity in a TRPA1-dependent manner ( Figure 3G-I ).…”

Investigation of Cholecystokinin-Beta receptor, IL-27, IL-27 gene SNP and some biochemical parameters in patients with Type-1 Diabetes Mellitus

“…Physiological CCK-AR stimulation induces exocrine pancreatic enzyme secretion and endocrine pancreatic polypeptide secretion 18 . CCK-AR limits acid secretion by releasing pepsin from primary gastric cells and somatic statin from gastric mucus D cells 19 . A decrease in small bowel passage time and a rise in colonic passage time are all caused by CCK-AR on smooth muscle cells, and the second is the anterior nucleus accumbens CCK-BR inhibits dopamine release.…”