Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.

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The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. In vivo, pancreatic protein output stimulated by CCK‐8 (400 pmol kg−1 h−1) was dose‐dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg−1 h−1) by 45 ± 8 and 84 ± 7 %, respectively. In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg−1 h−1) significantly inhibited CCK‐stimulated pancreatic protein secretion by 75 ± 18 %. Infusion of ghrelin (12 nmol kg−1 h−1) abolished pancreatic protein secretion caused by the central vagal stimulant 2‐deoxy‐d‐glucose (75 mg kg−1), whereas bethanechol‐stimulated pancreatic protein output was inhibited by only 59 ± 7 %. In vitro, ghrelin (10−11‐10−7m) produced no change in basal amylase release from dispersed, purified acinar cells. Co‐incubation of ghrelin (10−11−10−7m) with CCK−8 (10−10m) demonstrated no inhibition of CCK‐stimulated amylase release from dispersed acini. In contrast, ghrelin (10−9−10−7m) dose‐dependently inhibited amylase release from pancreatic lobules exposed to 75 mm potassium. Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons.

Intestinal barrier dysfunction and subsequent microbial translocation play crucial roles in persistent immune activation leading to HIV disease progression. Opioid use is associated with worse outcome in HIV-infected patients. The exacerbated disease progression by opioids is mainly driven by excessive intestinal inflammation and increased gut permeability. The objective of this study is to investigate how opioids potentiate HIV disease progression by compromising intestinal barrier function and impairing intestinal epithelial self-repair mechanism. In the present study, abnormal intestinal morphology and reduced epithelial proliferation were observed in bone marrow-liver-thymus humanized mice and in HIV-infected patients who were exposed to opioids. In bone marrow-liver-thymus mice, HIV, and morphine independently, and additively induced gut dysbiosis, especially depletion of Lachnospiraceae, Ruminococcaceae, and Muribaculaceae. We also observed that the abundance of Lachnospiraceae, Ruminococcaceae, and Muribaculaceae negatively correlated with apoptosis of epithelial cells, and intestinal IL-6 levels. Previous studies have shown that these bacterial families play crucial roles in maintaining intestinal homeostasis because they include most short-chain fatty acid-producing members. Short-chain fatty acids have been shown to maintain stem cell populations and suppress inflammation in the gut by inhibiting histone deacetylases (HDAC). In addition, we demonstrate that morphine exposure inhibited growth of intestinal organoids derived from HIV transgenic mice by suppressing Notch signaling in an HDAC-dependent manner. These studies implicate an important role for HDAC in intestinal homeostasis and supports HDAC modulation as a therapeutic intervention in improving care of HIV patients, especially in opioid-abusing population.

Abstract Background and Aims Opioids are the most prescribed analgesics for pain in inflammatory bowel diseases [IBD]; however, the consequences of opioid use on IBD severity are not well defined. This is the first study investigating consequences of hydromorphone in both dextran sodium sulphate [DSS]-induced colitis and spontaneous colitis (IL-10 knockout [IL-10-/-]) mouse models of IBD. Methods To determine the consequences of opioids on IBD pathogenesis, wild-type [WT] mice were treated with clinically relevant doses of hydromorphone and colitis was induced via 3% DSS in drinking water for 5 days. In parallel we also determined the consequences of opioids in a spontaneous colitis model. Results Hydromorphone and DSS independently induced barrier dysfunction, bacterial translocation, disruption of tight junction organisation and increased intestinal and systemic inflammation, which were exacerbated in mice receiving hydromorphone in combination with DSS. Hydromorphone + DSS-treated mice exhibited significant microbial dysbiosis. Predictive metagenomic analysis of the gut microbiota revealed high abundance in the bacterial communities associated with virulence, antibiotic resistance, toxin production, and inflammatory properties. Hydromorphone modulates tight junction organisation in a myosin light chain kinase [MLCK]-dependent manner. Treatment with MLCK inhibitor ML-7 ameliorates the detrimental effects of hydromorphone on DSS-induced colitis and thus decreases severity of IBD. Similarly, we demonstrated that hydromorphone treatment in IL-10-/- mice resulted in accelerated clinical manifestations of colitis compared with control mice. Conclusions Opioids used for pain management in IBD accelerate IBD progression by dysregulation of the gut microbiota, leading to expansion of pathogenic bacteria, translocation of bacteria, immune deregulation and sustained inflammation.