VPAC1 receptors regulate intestinal secretion and muscle contractility by activating cholinergic neurons in guinea pig jejunum.

In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally mediated and direct actions on smooth muscle and secretion by a direct mucosal action, and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors; however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin(+) excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY(+) secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 µM), PG97-269 (VPAC1 antagonist; 1 µM), and hyoscine (10 µM), but not by hexamethonium (200 µM). VIP (50 nM)-evoked secretion was depressed by hyoscine or PG97-269 and involved a small TTX-sensitive component. PG97-269 and TTX combined did not further depress the VIP response observed in the presence of PG97-269 alone. We conclude that VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl(-) secretion directly via epithelial VPAC1R and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.

Cholinergic Submucosal Neurons Display Increased Excitability Following in Vivo Cholera Toxin Exposure in Mouse Ileum.

Cholera-induced hypersecretion causes dehydration and death if untreated. Cholera toxin (CT) partly acts via the enteric nervous system (ENS) and induces long-lasting changes to enteric neuronal excitability following initial exposure, but the specific circuitry involved remains unclear. We examined this by first incubating CT or saline (control) in mouse ileal loops in vivo for 3.5 h and then assessed neuronal excitability in vitro using Ca2+ imaging and immunolabeling for the activity-dependent markers cFos and pCREB. Mice from a C57BL6 background, including Wnt1-Cre;R26R-GCaMP3 mice which express the fluorescent Ca2+ indicator GCaMP3 in its ENS, were used. Ca2+-imaging using this mouse model is a robust, high-throughput method which allowed us to examine the activity of numerous enteric neurons simultaneously and post-hoc immunohistochemistry enabled the neurochemical identification of the active neurons. Together, this provided novel insight into the CT-affected circuitry that was previously impossible to attain at such an accelerated pace. Ussing chamber measurements of electrogenic ion secretion showed that CT-treated preparations had higher basal secretion than controls. Recordings of Ca2+ activity from the submucous plexus showed that increased numbers of neurons were spontaneously active in CT-incubated tissue (control: 4/149; CT: 32/160; Fisher's exact test, P < 0.0001) and that cholinergic neurons were more responsive to electrical (single pulse and train of 20 pulses) or nicotinic (1,1-dimethyl-4-phenylpiperazinium (DMPP; 10 µM) stimulation. Expression of the neuronal activity marker, pCREB, was also increased in the CT-treated submucous plexus neurons. c-Fos expression and spontaneous fast excitatory postsynaptic potentials (EPSPs), recorded by intracellular electrodes, were increased by CT exposure in a small subset of myenteric neurons. However, the effect of CT on the myenteric plexus is less clear as spontaneous Ca2+ activity and electrical- or nicotinic-evoked Ca2+ responses were reduced. Thus, in a model where CT exposure evokes hypersecretion, we observed sustained activation of cholinergic secretomotor neuron activity in the submucous plexus, pointing to involvement of these neurons in the overall response to CT.

RCAN1 regulates mitochondrial function and increases susceptibility to oxidative stress in mammalian cells.

Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1(ox)) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12(RCAN1)). Similar to RCAN1(ox) neurons, PC12(RCAN1) cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.