Xenin-25 increases cytosolic free calcium levels and acetylcholine release from a subset of myenteric neurons

Xenin-25 (Xen) is a 25 amino acid neurotensin-related peptide reportedly produced with glucose-dependent insulinotropic polypeptide (GIP) by a subset of K cells in the proximal gut. We previously showed exogenously administered Xen, with GIP but not alone, increases insulin secretion in humans and mice. In mice, this effect is indirectly mediated via a central nervous system-independent cholinergic relay in the periphery. Xen also delays gastric emptying, reduces food intake, induces gall bladder contractions, and increases gut motility and secretion from the exocrine pancreas, suggesting that some effects of Xen could be mediated by myenteric neurons (MENs). To determine whether Xen activates these neurons, MENs were isolated from guinea pig proximal small intestines. Cells expressed neuronal markers and exhibited typical neuron-like morphology with extensive outgrowths emanating from cell bodies. Cytosolic free Ca²⁺ levels ([Ca²⁺]_i) were measured using Fura-2. ATP/UTP, KCl, and forskolin increased [Ca²⁺]_i in 99.6%, 92%, and 23% of the MENs imaged, respectively, indicating that they are functional and activated by nucleotide receptor signaling, direct depolarization, and cAMP. [Ca²⁺]_i increased in only 12.7% of MENs treated with Xen. This rise was blocked by pretreatment with EGTA, diazoxide, SR48692, and neurotensin. Thus the Xen-mediated increase in [Ca²⁺]_i involves influx of extracellular Ca²⁺ and activation of neurotensin receptor-1 (NTSR1). Xen also increased acetylcholine release from MENs. Amylin, produced by β-and enteroendocrine cells, delays gastric emptying and increased [Ca²⁺]_i almost exclusively in Xen-responsive MENs. Immunohistochemistry demonstrated NTSR1 expression in human duodenal MENs. Thus myenteric rather than central neurons could mediate some effects of Xen and amylin.